U.S. patent application number 11/523507 was filed with the patent office on 2007-08-09 for beta-secretase substrates and uses thereof.
Invention is credited to Stephen F. Brady, James E. Bruce, Elizabeth Chen-Dodson, Victor Garsky, Yueming Li, Mohinder Sardana, Jules A. Shafer, Xiaoting Tang.
Application Number | 20070184488 11/523507 |
Document ID | / |
Family ID | 26967437 |
Filed Date | 2007-08-09 |
United States Patent
Application |
20070184488 |
Kind Code |
A1 |
Brady; Stephen F. ; et
al. |
August 9, 2007 |
Beta-secretase substrates and uses thereof
Abstract
The present invention provides synthetic .beta.-secretase
peptide substrates useful in various assays for measuring
.beta.-secretase activity. Antibodies that recognize the synthetic
substrates and uses of the antibodies in various assays are
disclosed. The herein disclosed peptide substrates are hydrolyzed
at rates substantially faster than the attendant Swedish mutant APP
from which the substrate sequences are derived.
Inventors: |
Brady; Stephen F.;
(Philadelphia, PA) ; Bruce; James E.;
(Schwenksville, PA) ; Chen-Dodson; Elizabeth;
(Souderton, PA) ; Garsky; Victor; (Blue Bell,
PA) ; Li; Yueming; (New York, NY) ; Sardana;
Mohinder; (Lansdale, PA) ; Shafer; Jules A.;
(Gwynedd Valley, PA) ; Tang; Xiaoting; (Lansdale,
PA) |
Correspondence
Address: |
MERCK AND CO., INC
P O BOX 2000
RAHWAY
NJ
07065-0907
US
|
Family ID: |
26967437 |
Appl. No.: |
11/523507 |
Filed: |
September 19, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10480954 |
Jul 22, 2004 |
7132401 |
|
|
PCT/US02/15590 |
May 17, 2002 |
|
|
|
11523507 |
Sep 19, 2006 |
|
|
|
60292591 |
May 22, 2001 |
|
|
|
60316115 |
Aug 30, 2001 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
530/388.26 |
Current CPC
Class: |
C07K 7/06 20130101; C12Q
1/37 20130101; G01N 33/6896 20130101; G01N 2500/00 20130101; C12P
21/06 20130101; C07K 14/4711 20130101 |
Class at
Publication: |
435/007.1 ;
530/388.26 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/40 20060101 C07K016/40 |
Claims
1-18. (canceled)
19. A method for assaying for decreased .beta.-secretase activity
comprising measuring a cleavage product produced from cleavage by
.beta.-secretase of an APP substrate comprising a .beta.-secretase
peptide substrate comprising a .beta.-secretase cleavage site and
having the formula P4-P3-P2-P1-P1'-P2'-P3'-P4', where P4 is
selected from the group consisting of D, E, I, L and V; P3 is
selected from the group consisting of L, I, V and N; P2 is selected
from the group consisting of F, Y, E and N; P1 is selected from the
group consisting of F, I, L and Y; P1' is selected from the group
consisting of A, E, S, I, L and D; P2' is selected from the group
consisting of V, I, L and A; P3' is selected from the group
consisting of V, I, L, E and Y; and P4' is selected from the group
consisting of P, W and F; where P4-P3-P2-P1-P1'-P2'-P3'-P4' cannot
simultaneously be chosen such that they comprise the Swedish
mutation .beta.-secretase cleavage site (SEQ ID NO: 257); and where
a decrease in .beta.-secretase activity is determined when the
amount of the cleavage product is decreased relative to the amount
of the cleavage product produced from an APP substrate lacking said
.beta.-secretase cleavage site.
20. The method of claim 19 wherein said .beta.-secretase peptide
substrate corresponds to positions 593-600 of APP695.
21. The method of claim 20 wherein said .beta.-secretase peptide
substrate comprises SEQ ID NO: 262.
22. The method of claim 19 wherein the .beta.-secretase activity is
measured using an antibody that binds to the carboxyl terminus of
the cleaved APPsubstrate.
23. The method of claim 19 wherein the cleavage product detected is
the C-terminal cleavage product of said APP substrate.
24. The method of claim 19 for assaying for decreased
.beta.-secretase activity in the presence of one or more test
compounds, where the decreased .beta.-secretase activity is
determined when the amount of the cleavage product is decreased
relative to the amount of the cleavage product produced from an APP
substrate in the absence of said compounds.
25. The method of claim 24 wherein the decreased .beta.-secretase
activity in the presence of one or more test compounds is
indicative that said compounds inhibit .beta.-secretase
activity.
26. The method of claim 24 wherein said .beta.-secretase peptide
substrate corresponds to positions 593-600 of APP695.
27. The method of claim 24 wherein said .beta.-secretase substrate
comprises SEQ ID NO: 262.
28. The method of claim 24 wherein the .beta.-secretase activity is
measured using an antibody that binds to the carboxyl terminus of
the cleaved APPsubstrate.
29. The method of claim 19 wherein the cleavage product detected is
C-terminal cleavage product of said APP substrate.
30. An antibody that specifically binds to a cleavage product
produced from the cleavage by .beta.-secretase of an APP substrate
comprising a .beta.-secretase peptide substrate comprising a
.beta.-secretase cleavage site of claim 19.
31. An antibody of claim 30 wherein said antibody is a monoclonal
antibody.
32. An antibody of claim 30 wherein said antibody is a humanized
antibody.
33. An antibody of claim 30 that specifically binds to an
N-terminal cleavage product.
34. An antibody of claim 30 that specifically binds to a C-terminal
cleavage product.
35. An antibody of claim 30 that specifically binds to the
.beta.-secretase cleavage site corresponding to amino acids 596 and
597 numbered according to the APP695 isoform.
36. An antibody of claim 30 characterized by its ability to be
cleaved by human .beta.-secretase at a site corresponding to amino
acids 596 and 597 numbered according to the APP695 isoform.
Description
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
[0001] Not applicable.
REFERENCE TO MICROFICHE APPENDIX
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of
neurological and physiological dysfunctions associated with
Alzheimer's Disease. More particularly, the invention is concerned
with the identification of peptides that act as substrates for
.beta.-secretase. The present invention also relates to methods of
inhibiting the activity of .beta.-secretase as well as to methods
of screening for potential therapeutics for Alzheimer's Disease and
to methods of treatment for Alzheimer's Disease. Methods for
identifying compounds that modulate the activity of
.beta.-secretase are also provided.
BACKGROUND OF THE INVENTION
[0004] Few subjects in medicine today arouse the interest of the
scientific community and the lay community as does Alzheimer's
disease (AD). AD has emerged as the most prevalent form of
late-life mental failure in humans. AD is a common dementing brain
disorder of the elderly. The key features of the disease include
progressive memory impairment, loss of language and visuospatial
skills, and behavior deficits. These changes in cognitive function
are the result of degeneration of neurons in the cerebral cortex,
hippocampus, basal forebrain, and other regions of the brain.
Neuropathological analyses of postmortem Alzheimer's diseased
brains consistently reveal the presence of large numbers of
neurofibrillary tangles in degenerated neurons and neuritic plaques
in the extracellular space and in the walls of the cerebral
microvasculature. The neurofibrillary tangles are composed of
bundles of paired helical filaments containing hyperphosphorylated
tau protein (Lee & Trojanowski, 1992, Curr. Opin. Neurobiol.
2:653-656). The neuritic plaques consist of deposits of
proteinaceous material surrounding an amyloid core (Selkoe, 1994,
Annu. Rev. Neurosci. 17:489-517).
[0005] AD has been estimated to affect more than 4 million people
in the United States alone and perhaps 17 to 25 million worldwide.
Moreover, the number of sufferers is expected to grow as the
population ages. The pathology of AD has been studied extensively
for the last 20 years, but it was not until about 15 years ago that
the first molecular handle in understanding this complex
degenerative disease was obtained, when the protein sequence of the
extracellular amyloid was determined.
[0006] The effort to decipher the mechanism of AD has attracted the
interest of investigators from diverse biological disciplines,
including biochemistry, cell biology, molecular genetics,
neuroscience, and structural biology. The eclectic nature of
research approaches to AD and the intensity of scientific interest
in the problem have made it increasingly likely that AD will become
a premier example of the successful application of biological
chemistry to the identification of rational therapeutic targets in
a major human disease. Much of the recent progress in elucidating
the pathogenesis of AD has centered on the apparent role of the
40-42-residue amyloid-protein (A.beta.) as a unifying pathological
feature of the genetically diverse forms of this complex
disorder.
[0007] AD is divided into 2 classes: Familial AD, (FAD) which has
an early onset and is heritable, and "non-familial", or sporadic AD
(SAD), which has no identifiable cause. Although FAD is rare (less
than 10% of all AD), the characteristic clinicopathological
features--amyloid plaques, neurofibrillary tangles, synaptic and
neuronal loss, and neurotransmitter deficits are apparently
indistinguishable from the more common SAD.
[0008] The defining neuropathological characteristic of AD is the
accumulation of insoluble proteinacious deposits, known as amyloid
plaques, in the brains of those affected. The presence of these
amyloid plaque deposits is the essential observation underpinning
the amyloid hypothesis.
[0009] Evidence suggests that deposition of amyloid-.beta. peptide
(AP) plays a significant role in the development of amyloid plaques
and the etiology of AD. For example, individuals with mutations in
the gene encoding the .beta.-amyloid precursor protein (APP) from
which the A.beta. protein is derived invariably develop Alzheimer's
disease (Goate et al., 1991, Nature 353:844-846; Muilan et al.,
1992, Nature Genet. 1:345-347; Murrell et al., 1991, Science
254:97-99; Van Broeckhoven, 1995, Eur. J. Neurol. 35:8-19).
Likewise, autopsies have shown that amyloid plaques are found in
the brains of virtually all Alzheimer's patients and that the
degree of amyloid plaque deposition correlates with the degree of
dementia (Cummings & Cotman, 1995, Lancet 326:1524-1587).
[0010] That increased expression and/or abnormal processing of APP
is associated with the formation of amyloid plaques and
cerebrovascular amyloid deposits, which are one of the major
morphological hallmarks of AD has been corroborated from least two
sources. The first is that transgenic mice which express altered
APP genes exhibit neuritic plaques and age-dependent memory
deficits (Games et al., 1995, Nature 373:523-525; Masliah et al.,
1996, J. Neurosci. 16:5795-5811; Hsiao et al., 1996, Science
274:99-103).
[0011] The second body of evidence comes from study of patients
suffering from Down's syndrome, who develop amyloid plaques and
other symptoms of Alzheimer's disease at an early age (Mann &
Esiri, 1989, J. Neurosci. 89:169-179). Because the APP gene is
found on chromosome 21, it has been hypothesized that the increased
gene dosage which results from the extra copy of this chromosome in
Down's syndrome accounts for the early appearance of amyloid
plaques (Kang et al., 1987, Nature 325:733-736; Tanzi et al., 1987,
Science 235:880-884). Taken together with the evidence derived from
cases of familial Alzheimer's disease, the current data suggest
that genetic alterations which result in an increase in A.beta.
production can induce Alzheimer's disease. Accordingly, since
A.beta. deposition is an early and invariant event in Alzheimer's
disease, it is believed that treatment which reduces production of
A.beta. will be useful in the treatment of this disease. Among the
processes regulating APP metabolism, the proteolytic cleavage of
APP into amyloidogenic or nonamyloidogenic fragments is of special
interest.
[0012] The strongest evidence implicating A.beta. in the
pathogenesis of AD comes from the observation that A.beta. peptides
are toxic to neurons in culture and transgenic mice that
overproduce A.beta. in their brains show significant deposition of
A.beta. into amyloid plaques and significant neuronal toxicity
(Yankner et al., 1989, Science 245:417-420; Frautschy et al., 1991,
Proc. Nati. Acad. Sci. USA 88:8362-8366; Kowall et al., 1991, Proc.
Natl. Acad. Sci. USA 88:7247-7251). This toxicity is enhanced if
the peptides are "aged" (incubated from hours to days), a procedure
that increases amyloid fibril formation. As well, injection of the
insoluble, fibrillar form of A.beta. into monkey brains results in
the development of pathology (neuronal destruction, tau
phosphorylation, microglial proliferation) that closely mimics
Alzheimer's disease in humans (Geula et al., 1998, Nature Medicine
4:827-831). See Selkoe, 1994, J. Neuropathol. Exp. Neurol.
53:438-447 for a review of the evidence that amyloid plaques have a
central role in Alzheimer's disease.
[0013] While abundant evidence suggests that extracellular
accumulation and deposition of A.beta. is a central event in the
etiology of AD, recent studies have also proposed that increased
intracellular accumulation of A.beta. or amyloid containing
C-terminal fragments may play a role in the pathophysiology of AD.
For example, over-expression of APP harboring mutations which cause
familial AD results in the increased intracellular accumulation of
C100 in neuronal cultures and A.beta.42 in HEK 293 cells. A.beta.42
is the 42 amino acid long form of A.beta. that is believed to be
more efficacious at formed amyloid plaques than shorter forms of
A.beta.. Moreover, evidence suggests that intra- and extracellular
A.beta. are formed in distinct cellular pools in hippocampal
neurons and that a common feature associated with two types of
familial AD mutations in APP ("Swedish" and "London") is an
increased intracellular accumulation of A.beta.42. Thus, based on
these studies and earlier reports implicating extracellular A.beta.
accumulation in AD pathology, it appears that altered APP
catabolism may be involved in disease progression.
[0014] APP is an ubiquitous membrane-spanning (type 1) glycoprotein
that undergoes a variety of proteolytic processing events. (Selkoe,
1998, Trends Cell Biol. 8:447-453). APP is actually a family of
peptides produced by alternative splicing from a single gene. Major
forms of APP are known as APP.sub.695, APP.sub.751, and
APP.sub.770, with the subscripts referring to the number of amino
acids in each splice variant (Ponte et al., 1988, Nature
331:525-527; Tanzi et al., 1988, Nature 331:528-530; Kitaguchi et
al., 1988, Nature 331:530-532 ). APP is expressed and
constitutively catabolized in most cells.
[0015] APP has a short half-life and is metabolized rapidly down
two pathways in all cells. The dominant catabolic pathway appears
to be cleavage of APP within the A.beta. sequence by
.alpha.-secretase, resulting in the constitutive secretion of a
soluble extracellular domain (sAPP.alpha.) and the appearance of a
nonamyloidogenic intracellular fragment (approximately 9 kD),
referred to as the constitutive carboxy-terminal fragment
(cCTF.alpha.). cCTF.alpha. is a suitable substrate for cleavage by
.gamma.-secretase to yield the p3 fragment. This pathway appears to
be widely conserved among species and present in many cell types
(Weidemann et al., 1989, Cell 57:115-126; Oltersdorf et al., 1990,
J. Biol. Chem. 265:4492-4497; and Esch et al., 1990, Science
248:1122-1124). In this pathway, processing of APP involves
proteolytic cleavage at a site between residues Lys.sub.16 and
Leu.sub.17 of the A.beta. region while APP is still in the
trans-Golgi secretory compartment (Kang et al., 1987, Nature
325:773-776). Since this cleavage occurs within the A.beta. portion
of APP, it precludes the formation of A.beta.. sAPP.alpha. has
neurotrophic and neuroprotective activities (Kuentzel et al., 1993,
Biochem. J. 295:367-378).
[0016] In contrast to this non-amyloidogenic pathway involving
.alpha.-secretase described above, proteolytic processing of APP by
.beta.-secretase exposes the N-terminus of A.beta., which after
.gamma.-secretase cleavage at the variable C-terminus, liberates
A.beta.. This A.beta.-producing pathway involves cleavage of the
Met.sub.671-ASp.sub.672 bond (numbered according to the 770 amino
acid isoform) by .beta.-secretase. The C-terminus is actually a
heterogeneous collection of cleavage sites rather than a single
site since .gamma.-secretase activity occurs over a short stretch
of APP amino acids rather than at a single peptide bond. In the
amyloidogenic pathway, APP is cleaved by .beta.-secretase to
liberate sAPP.beta. and CTF.beta., which CTF.beta. is then cleaved
by .gamma.-secretase to liberate the harmful A.beta. peptide.
[0017] Of key importance in this A.beta.-producing pathway is the
position of the .gamma.-secretase cleavage. If the
.gamma.-secretase cut is at residue 711-712, short A.beta.
(A.beta.40) is the result; if it is cut after residue 713, long
A.beta. (A.beta.42) is the result. Thus, the .gamma.-secretase
process is central to the production of A.beta. peptide of 40 or 42
amino acids in length (A.beta.40 and A.beta.42, respectively). For
a review that discusses APP and its processing, see Selkoe, 1998,
Trends Cell. Biol. 8:447453; Selkoe, 1994, Ann. Rev. Cell Biol.
10:373-403. See also, Esch et al., 1994, Science 248:1122.
[0018] A.beta., the principal component of amyloid plaques, is a
39-43 aminio acid peptide which is capable of forming
.beta.-pleated sheet aggregates. These aggregating fibrils are
subsequently deposited in the brain parenchyma or in the
cerebrovasculature of the Alzheimer's disease victim (Glenner et
al., 1984, Biochem. Biophys. Res. Comm. 120:885-890; Masters et
al., 1985, Proc. Natl. Acad. Sci. USA 82:42454249).
[0019] Reports show that soluble .beta.-amyloid peptide is produced
by healthy cells into culture media (Haass et al., 1992, Nature
359:322-325) and in human and animal CSF (Seubert et al., 1992,
Nature 359:325-327).
[0020] Cleavage of APP can be detected in a number of convenient
manners, including the detection of polypeptide or peptide
fragments produced by proteolysis. Such fragments can be detected
by any convenient means, such as by antibody binding. Another
convenient method for detecting proteolytic cleavage is through the
use of a chromogenic .beta.-secretase substrate whereby cleavage of
the substrate releases a chromogen, e.g., a colored or fluorescent,
product.
[0021] Various groups have cloned and sequenced cDNA encoding a
protein that is believed to be .beta.-secretase (Vassar et al.,
1999, Science 286:735-741; Hussain et al., 1999, Mol. Cell.
Neurosci. 14:419-427; Yan et al., 1999, Nature 402:533-537; Sinha
et al., 1999, Nature 402:537-540; Lin et al., 2000, Proc. NatI.
Acad. Sci. USA 97:1456-1460). .beta.-secretase has been called
various names by these groups, e.g., BACE, Asp2, memapsin2.
[0022] Much interest has focused on the possibility of inhibiting
the development of amyloid plaques as a means of preventing or
ameliorating the symptoms of Alzheimer's disease. To that end, a
promising strategy is to inhibit the activity of at least one of
.beta.- and .gamma.-secretase, the two enzymes that together are
responsible for producing A.beta.. This strategy is attractive
because, if the formation of amyloid plaques as a result of the
deposition of A.beta. is a cause of Alzheimer's disease, inhibiting
the activity of one or both of the two secretases would intervene
in the disease process at an early stage, before late-stage events
such as inflammation or apoptosis occur. Such early stage
intervention is expected to be particularly beneficial (see, e.g.,
Citron, 2000, Molecular Medicine Today 6:392-397).
[0023] Thus, it is believed that a drug that could interfere with
.beta.-amyloid plaque formation or toxicity may delay or halt the
progression of Alzheimer's disease. At present, few suitable in
vitro systems or methods exist for screening candidate drugs for
the ability to inhibit or prevent the production of .beta.-amyloid
plaque. The scarcity of such screening methods may, at least in
part, result from insufficient understanding of the pathogenic
mechanism(s) which cause the conversion of amyloid precursor
protein to the .beta.-amyloid peptide, and ultimately to the
amyloid plaque.
[0024] In view of the anticipated benefits of modulating APP
catabolism as a treatment for diseases such as AD, compositions and
methods for modulating APP catabolism in APP-containing cells which
do not substantially alter the viability of those cells, have been
desired and are addressed by the present invention.
[0025] For these reasons, it would be desirable to provide methods
and systems for screening test compounds for the ability to inhibit
or prevent the production of A.beta. from APP. In particular, it
would be desirable to base such methods and systems on a metabolic
pathway which is involved in such conversion, where the test
compound would be able to interrupt or interfere with the metabolic
pathway which leads to conversion. In particular, initial methods
should utilize in vitro systems rather than animal models, so that
the methods are particularly suitable for initial screening of test
compounds to identify suitable candidate drugs.
SUMMARY OF THE INVENTION
[0026] The present invention features 8-mer peptides that are
suitable substrates for .beta.-secretase, particularly human
.beta.-secretase. In vitro assays for measuring .beta.-secretase
activity employing such substrates are also disclosed. The peptide
substrates are characterized as comprising at least one
.beta.-secretase cleavage site.
[0027] In one embodiment, the peptide substrates of the invention,
when presented as an immunogen, elicit the production of a
antibodies which specifically bind to a region of native APP having
an amino acid sequence that is substantially homologous to that of
any of the disclosed invention peptides. In another embodiment, the
antibodies specifically bind the peptide substrates of the
invention but do not specifically bind to native APP.
[0028] In another aspect of the present invention, antibodies are
provided which are specific for an amino terminal fragment or
carboxy terminal fragment (a/k/a cleavage product) of one of the
peptides listed in Table 1 and/or Table 2 that results from
cleavage of the substrates by .beta.-secretase. The antibodies may
be polyclonal or monoclonal.
[0029] In another aspect, the invention provides antibodies that
recognize the synthetic .beta.-secretase cleavage site of any of
the 8-mer peptides listed in Table 1 and/or Table 2. In particular,
antibodies that do not recognize the .beta.-secretase cleavage site
of native APP are provided.
[0030] An aspect of the present invention describes a nucleic acid
comprising a nucleotide base sequence encoding any of the herein
disclosed .beta.-secretase substrates. Preferably, the nucleic acid
is contained in an expression vector.
[0031] Another aspect of the present invention describes a
recombinant cell comprising a nucleic acid encoding any of the
disclosed .beta.-secretase substrates or fragments thereof.
[0032] Another aspect of the present invention describes a method
for assaying .beta.-secretase activity. .beta.-Secretase activity
can be obtained from cells producing .beta.-secretase in a
solubilized form or in a membrane-bound form. The method can be
performed by measuring cleavage product formation resulting from
.beta.-secretase substrate cleavage. Measuring can be performed by
qualitative or quantitative techniques.
[0033] Thus an aspect of the present invention describes a method
for measuring the ability of a compound to affect .beta.-secretase
activity comprising the steps of: (a) combining together a
.beta.-secretase substrate, a test compound, and a preparation
comprising .beta.-secretase activity, under reaction conditions
allowing for .beta.-secretase activity, and (b) measuring
.beta.-secretase activity.
[0034] An exemplary method utilizes a reaction system including
.beta.-secretase and a peptide substrate of the invention present
in initial amounts. The reaction system is maintained under
conditions which permit the .beta.-secretase to cleave the peptide
substrate into cleavage products. The .beta.-secretase cleavage
reaction is monitored by detecting the amount of at least one of
the .beta.-secretase cleavage products, where the amount of
cleavage product(s) will increase over time as the reaction
progresses. Such methods are particularly useful for screening test
compounds for the ability to inhibit .beta.-secretase activity.
Test compounds are introduced to the reaction system, and the
ability of the test compound to inhibit the .beta.-secretase
activity is determined based on the ability to decrease the amount
of cleavage product produced, usually in comparison to a control
where .beta.-secretase mediated cleavage in the reaction system is
observed and measured in the absence of test compound(s).
[0035] The methods of the present invention allow identification of
test substances which inhibit proteolytic cleavage of to disclosed
peptide substrates by .beta.-secretase. The methods comprise
exposing a peptide of the invention comprising a .beta.-secretase
cleavage site to .beta.-secretase in the presence of the test
substance under conditions such that the .beta.-secretase would be
expected to cleave the peptide substrate into an amino-terminal
fragment and a carboxy-terminal fragment in the absence of the test
substance. Test substances which inhibit such cleavage are thus
identified as having .beta.-secretase inhibition activity. Usually,
generation of the amino-terminal fragment and/or the
carboxy-terminal fragment is detected by an antibody specific for
the carboxy end of the amino-terminal fragment or the amino end of
the carboxy-terminal fragment. Alternative methods of detecting the
amino-terminal and/or carboxyl-terminal fragments include liquid
chromatography/mass spectrometry (LC/MS).
[0036] The present invention further comprises methods for
inhibiting the cleavage of .beta.-amyloid substrate in cells. Such
methods comprise administering to the cells an amount of a compound
effective to at least partially inhibit .beta.-secretase activity.
Usually, such compounds will be selected by the screening methods
described above. Such compounds, will also find use in inhibiting
binding of native or recombinant .beta.-secretase to APP in
vivo.
[0037] The present invention further provides methods for
inhibiting the cleavage of .beta.-amyloid precursor protein in
mammalian hosts. Such methods comprise administering to the host an
amount of a compound effective to inhibit .beta.-secretase activity
in cells of the host, usually in brain cells of the host. Such
compounds will usually be selected by the screening assays
described in this application. Such methods will be useful for
treating conditions related to A.beta. peptide deposition such as
Alzheimer's disease, Down's syndrome, and the like.
[0038] In another aspect of the present invention, A.beta.
production inhibitors identified by the methods described herein
may be studied in transgenic animal models. The animals are exposed
to test compound(s) and those compounds which affect (usually by
diminishing) the production of any of the cleavage products of APP
are considered candidates for further testing as drugs for the
treatment of A.beta.-related conditions.
[0039] Methods and compositions are provided for detecting and
monitoring an amino-terminal fragment resulting from
.beta.-secretase cleavage of any of the herein included peptide
substrates. The resulting fragment, referred to as a an amino
terminal cleavage product, .beta.ATF-PS (amino terminal fragment of
the peptide substrates) may be detected in biological samples and
is useful for monitoring the processing of the disclose substrates
in animal models. In particular, the present invention provides for
monitoring in vivo processing of any of the disclosed peptide
substrates where the presence of the .beta.ATF-PS is detected in a
specimen from an animal transformed to express the substrates and
where the .beta.ATF-PS has been cleaved from the substrate between
amino acids 596 and 597, based on the numbering of Kang et al.,
1987, Nature 325:733-736 in the 695 amino acid isoform.
[0040] It has been found that cells expressing the gene encoding
any of the herein disclosed peptide substrates are particularly
prolific producers of the cleavage products of said peptide
substrate that are the amino- and the carboxy-terminal fragments
thereof. That is, such cells are able to cleave the substrates of
the invention at a greater frequency than cleavage of either the
endogenous APP, the wild type human APP, or the Swedish mutant APP.
It is further believed that intracellular processing of the
substrates of the invention results in greater production of the
.beta.ATF-PS than is produced by other human mutations of the APP
gene. Thus, transgenic animal systems, such as transgenic mice,
expressing any of the herein disclosed peptide substrates are
particularly suitable as models for monitoring intracellular
processing of the disclosed substrates as well as for screening
test compounds for the ability to inhibit or modulate cleavage of
APP as a result of .beta.-secretase activity, the apparently
pathogenic form of APP processing.
[0041] Other features and advantages of the present invention are
apparent from the additional descriptions provided herein including
the different examples. The provided examples illustrate different
components and methodology useful in practicing the present
invention. The examples do not limit the claimed invention. Based
on the present disclosure the skilled artisan can identify and
employ other components and methodology useful for practicing the
present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 depicts peptide libraries derived from Swedish mutant
APP. The amino acids are represented in single letter code. SEQ ID
NO: 257 (top row) represents Swedish mutant APP corresponding to
amino acids 593-560 based upon numbering of the 695 isoform of
APP.
[0043] FIG. 2 depicts data illustrating the differences in rates of
cleavage of the various peptide substrates from the P1 library by
.beta.-secretase.
[0044] FIG. 3 illustrates the relative preference of
.beta.-secretase for a specified amino acid based upon an analysis
of a cleavage product (C-terminal fragment) formed after cleavage
of the invention peptide substrate(s) derived from the P1' peptide
library by the .beta.-secretase.
[0045] FIG. 4 depicts a graph showing the most preferred residues
for a peptide substrate sequence based upon the rate of hydrolysis
of a substrate by .beta.-secretase. The data show the preferred
sequence at specific positions of an 8-mer sequence that increases
the rate of hydrolysis of the substrate sequence by
.beta.-secretase.
[0046] FIG. 5 depicts the increase in the rate of hydrolysis of
synthetic 8-mer peptide substrates observed with the incorporation
of amino acid substitutions for positions P1 and P1' based on the
preference for amino acids Phe and Ala, respectively. The
replacement of Ala for Asp 597 in an 8-mer peptide sequence
corresponding to the Swedish mutant sequence 695 resulted in an
increase in the observed rate of hydrolysis of 10 relative to the
Swedish mutant sequence. Additional incorporation of Phe for Leu
596 in an 8-mer peptide sequence resulted in an observed hydrolysis
by .beta.-secretase that was 17 times higher than that observed
with the Swedish mutant sequence.
[0047] FIG. 6 shows the rate of hydrolysis for the Swedish mutant
(EVNLDAEF; SEQ ID NO: 257) compared with the rate for the invention
peptides EVNFAAEF (SEQ ID NO: 259); EVNFEAEF (SEQ ID NO: 260);
EVNFAVEF (SEQ ID NO: 261); and EVNFEVEF (SEQ ID NO: 262).
DETAILED DESCRIPTION OF THE INVENTION
[0048] As used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. Thus, for example, reference to
"a host cell" includes a plurality of such host cells, reference to
"the antibody" is a reference to one or more antibodies and
equivalents thereof known to those skilled in the art, and so
forth.
[0049] The present invention features .beta.-secretase substrates
and assays for detecting .beta.-secretase activity employing such
substrates. The .beta.-secretase substrate can be cleaved by
.beta.-secretase activity.
[0050] Assaying for .beta.-secretase activity can be used, for
example, to screen for compounds able to modulate .beta.-secretase
activity, and to test the ability of a particular compound to
affect .beta.-secretase activity. Examples of compounds able to
modulate .beta.-secretase activity include .beta.-secretase
inhibitors. Inhibitors can be employed for different purposes, such
as in the treatment of Alzheimer's disease or characterization of
the biological importance of .beta.-secretase.
[0051] Provided herein are numerous synthetic 8-mer peptides, each
comprising a synthetic .beta.-secretase cleavage site. Table 2
lists 256 8-mer peptide substrate sequences that are substrates for
.beta.-secretase, particularly human .beta.-secretase.
Polynucleotides encoding the peptide substrate sequences and
methods employing the peptide substrate sequences in assays to
identify compounds that inhibit the proteolytic activity of
.beta.-secretase, native or recombinant, in vitro or in vivo, are
also within the scope of the present invention.
[0052] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described.
[0053] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the
methodologies, vectors, etc. which are reported in the publications
that might be used in connection with the invention. Nothing herein
is to be construed as an admission that the invention is not
entitled to antedate such disclosures by virtue of prior
invention.
[0054] In the description that follows, a number of terms used in
the field of recombinant DNA technology are extensively utilized.
In order to provide a clearer and consistent understanding of the
specification and claims, including the scope to be given such
terms, the following definitions are provided.
[0055] Use of the terms "isolated" and/or "purified" in the present
specification and claims as a modifier of DNA, RNA, polypeptides,
peptides, or proteins means that the DNA, RNA, polypeptides,
peptides, or proteins so designated have been produced in such form
by the hand of man, and thus are separated from their native in
vivo cellular environment. As a result of this human intervention,
the recombinant DNAs, RNAs, polypeptides, peptides, or proteins of
the invention are useful in ways described herein that the DNAs,
RNAs, polypeptides, peptides, or proteins as they naturally occur
are not. A peptide that has been produced by synthetic means, e.g.,
by the solid phase chemistry disclosed herein, and that is the
predominant peptide species present, is an "isolated" or "purified"
peptide.
[0056] Similarly, as used herein, "recombinant" as a modifier of
DNA, RNA, polypeptides, peptides, or proteins means that the DNA,
RNA, polypeptides, peptides, or proteins so designated have been
prepared by the efforts of human beings, e.g., by cloning,
recombinant expression, and the like. Thus as used herein,
recombinant proteins, for example, refers to proteins produced by a
recombinant host, expressing DNAs which have been added to that
host through the efforts of human beings.
[0057] An "insertion" or "addition," as used herein, refers to a
change in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid or nucleotide residues,
respectively, as compared to the naturally occurring molecule.
[0058] A "substitution", as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0059] A "disorder" is any condition that would benefit from
treatment with (1) the disclosed invention peptides (2) antibodies
specific for each of the disclosed invention peptides (3) or any
compound that inhibits the proteolytic activity of
.beta.-secretase. This includes chronic and acute disorders or
diseases including those pathological conditions which predispose
the mammal to the disorder in question. Disorders include
Alzheimer's Disease. "Antagonist" or "inhibitor" as used herein
refers to an agent that downregulates (e.g., suppresses or
inhibits) at least one .beta.-secretase bioactivity. A
.beta.-secretase inhibitor can be a compound or molecule which
inhibits or decreases the interaction between a .beta.-secretase
and its binding partner, e.g., native or mutant APP or any one of
the disclosed invention peptides. A compound that decreases the
rate at which .beta.-secretase cleaves its substrate is a
.beta.-secretase inhibitor.
[0060] The term "modulation" is used herein to refer to the
capacity to inhibit a biological or functional activity, e.g.,
proteolytic activity and/or pharmacological activity of human
.beta.-secretase. "Substantially similar" indicates a sequence
similarity of at least about 80% to a reference sequence. In
different embodiments the sequence similarity is at least about
90%, at least about 95% or 100%. Sequence similarity can be
determined using techniques well known in the art, such as those
described by Altschul et al., 1997, Nucleic Acids Res.
25:3389-3402, hereby incorporated by reference herein. "Direct
administration" as used herein refers to the direct administration
of antibodies specific for any of the herein disclosed invention
peptides or functional derivatives thereof that inhibit the
proteolytic activity of human .beta.-secretase. Compounds that
achieve an inhibitory effect are also included.
[0061] A ".beta.-CTF domain" is a polypeptide that can be cleaved
by .gamma.-secretase and which approximates the C-terminal fragment
(amino acids 597-695) of APP produced after cleavage of APP by a
.beta.-secretase, or is a functional derivative thereof.
[0062] As used herein, ".beta.-amyloid peptide (A.beta.)" refers to
any of the .beta.-amyloid peptides. Such peptides are typically
about 4 kD. Several different amino-termini and heterogeneous
carboxyl-termini sequences have been observed based on
characterization of A.beta. from Alzheimer's disease tissue and
from cultured cells (Glenner & Wong, 1984, Biochem. Biophys.
Res. Comm. 120:885-890; Joachim et al., 1988, Brain Res. 474:
100-111; Prelli et al., 1988, J. Neurochem. 51:648-651; Mori et
al., 1992, J. Biol. Chem. 267:17082-17806; Seubert et al., 1992,
Nature 359:325-327; Naslund et al., 1994, Proc. Natl. Acad. Sci.
USA 91:8378-8382; Roher et al., 1993, Proc. NatI. Acad. Sci. USA
90:10836-10840; Busciglio et al., 1993, Proc. Natl. Acad. Sci. USA
90:2092-2096; Haass et al., 1992, Nature 359:322-325). With regard
to the carboxyl-termini, A.beta. has been shown to end at position
39, 40, 41, 42, 43, or 44 where position 1 is the aspartate of the
A.beta. sequence of SEQ ID NO: 257. A.beta. is produced by the
processing of APP including cleavage at both the amino-terminus and
carboxy-terminus of the A.beta. region of APP.
[0063] As used herein, ".beta.-amyloid precursor protein (APP)"
refers to a protein that is encoded by a gene of the same name
localized in humans on the long arm of chromosome 21 and that
includes the A.beta. region within its carboxyl third. APP is a
glycosylated, single-membrane-spanning protein expressed in a wide
variety of cells in many mammalian tissues. Examples of specific
isotypes of APP which are currently known to exist in humans are
the 695-amino acid polypeptide described by Kang et al., 1987,
Nature 325:733-736; a 751-amino acid polypeptide described by Ponte
et al., 1988, Nature 331:525-527 and Tanzi et al., 1988, Nature
331:528-530; as well as a 770-amino acid isotype of APP described
in Kitaguchi et al., 1988, Nature 331:530-532. A number of specific
variants of APP have also been described having point mutations
which can differ in both position and phenotype. A general review
of such mutations is provided in Hardy, 1992, Nature Genet.
1:233-234. A mutation of particular interest is designated the
"Swedish" mutation where the normal Lys-Met residues at positions
595 and 596 of the 695 form are replaced by Asn-Leu. This mutation
is located directly upstream of the normal .beta.-secretase
cleavage site of APP, which occurs between residues 596 and 597 of
the 695 form. SEQ ID NO: 257 shows the amino acid sequence of the
Swedish mutant in the region of the .beta.-secretase cleavage
site.
[0064] As used herein, the term "human .beta.-secretase," and
".beta.-secretase" refers to a protein/enzyme, that exhibits the
same properties as that attending native .beta.-secretase, i.e.,
ability to cleave APP and yield the amino terminus of A.beta.
peptide that is the main component of the amyloid plaques found in
patients suffering from Alzheimer's disease.
[0065] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0066] The terms "recombinant protein" refer to a protein that is
produced by expression of a nucleotide sequence encoding the amino
acid sequence of the protein from a recombinant DNA molecule.
[0067] A "functional derivative thereof" has a sufficient sequence
similarity to the invention peptide amino acid sequences such that
it can be cleaved by .beta.-secretase. Examples of modifications to
the invention peptide substrates that can produce a functional
derivative include additions, deletions, and substitutions. The
effect of a particular modification can be measured using reaction
conditions described herein that allow for .beta.-secretase
cleavage of a .beta.-secretase substrate. Preferred modifications
do not cause a substantial decrease in activity.
[0068] Substitutions in the substrate not causing a substantial
decrease in activity can be initially designed taking into account
differences in naturally occurring amino acid R groups. An R group
effects different properties of the amino acid such as physical
size, charge, and hydrophobicity. Amino acids can be divided into
different groups as follows: neutral and hydrophobic (alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalaine, and
methionine); neutral and polar (glycine, serine, threonine,
tryosine, cysteine, asparagine, and glutamine); basic (lysine,
arginine, and histidine); and acidic (aspartic acid and glutamic
acid).
[0069] Generally, in substituting different amino acids, it is
preferable to exchange amino acids having similar properties.
Substituting different amino acids within a particular group, such
as substituting valine for leucine, arginine for lysine, and
asparagine for glutamine is likely to not cause a change in peptide
functioning.
[0070] Changes outside of different amino acid groups can also be
made. Preferably, such changes are made taking into account the
position of the amino acid to be substituted in the peptide. For
example, arginine can substitute more freely for nonpolar amino
acids in the interior of a polypeptide then glutamate because of
its long aliphatic side chain. (See, Ausubel, Current Protocols in
Molecular Biology, John Wiley, 1987-1998, Supplement 33, Appendix
1C).
[0071] An amino acid sequence or a nucleotide sequence is
"identical" to a reference sequence if the two sequences are the
same when aligned for maximum correspondence over a comparison
window. Optimal alignment of nucleotide and amino acid sequences
for aligning comparison window may be conducted by the local
homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math.
2:482, by the homology alignment algorithm of Needleman &
Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity
method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci.,
U.S.A. 85:2444-2448, by computerized implementations of these
algorithms (GAP, BESFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0072] "Consists essentially," with respect to a .beta.-secretase
substrate, indicates that the reference sequence can be modified by
N-terminal and/or C-terminal additions or deletions that do not
cause a substantial decrease in the ability of the .beta.-secretase
substrate to be cleaved compared to the reference sequence. An
example of a deletion is the removal of an N-terminal
methionine.
[0073] A substantial decrease in the ability of the
.beta.-secretase substrate to be cleaved is a decrease of 10 fold
or more compared to activity observed using a reference substrate
incubated with appropriate buffers and suitable reagents.
[0074] As used herein, "test compounds" may be any substance,
molecule, compound, mixture of molecules or compounds, or any other
composition which is suspected of being capable of inhibiting
.beta.-secretase activity in vivo or in vitro. The test compounds
may be macromolecules, such as biological polymers, including
proteins, polysaccharides, nucleic acids, or the like. More
usually, the test compounds will be small molecules having a
molecular weight below about 2 kD, more usually below 1.5 kD,
frequently below 1 kD, and usually in the range from 100 to 1,000
D, and even more usually in the range from 200 D to 750 D. Such
test compounds may be preselected based on a variety of criteria.
For example, suitable test compounds may be selected as having
known proteolytic inhibition activity. Alternatively, the test
compounds may be selected randomly and tested by the screening
methods of the present invention. Test compounds which are able to
inhibit .beta.-secretase cleavage of the invention peptide
substrates in vitro are considered as candidates for further
screening of their ability to decrease A.beta. production in cells
and/or animals.
[0075] The term "antibody" refers to a polypeptide substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof, which specifically bind and recognize an analyte
(antigen). The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon and mu constant region genes,
as well as the myriad immunoglobulin variable region genes.
Antibodies exist, e.g., as intact immunoglobulins or as a number of
well characterized fragments produced by digestion with various
peptidases. These include, e.g., Fab' and F(ab)'.sub.2 fragments.
The term "antibody" also includes antibody fragments either
produced by the modification of whole antibodies or those
synthesized de novo using recombinant DNA methodologies, and
further includes "humanized" antibodies made by conventional
techniques.
[0076] The term "immunoassay" is an assay that utilizes an antibody
to specifically bind an analyte. The immunoassay is characterized
by the use of specific binding properties of a particular antibody
to isolate, target, and/or quantify the analyte.
[0077] An antibody "specifically binds to" or "is specifically
immunoreactive with" a protein, polypeptide, or peptide when the
antibody functions in a binding reaction which is determinative of
the presence of the protein, polypeptide, or peptide in the
presence of a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind preferentially to a particular protein,
polypeptide, or peptide and do not bind in a significant amount to
other proteins, polpeptides, or peptides present in the sample.
Specific binding to a protein, polypeptide, or peptide under such
conditions requires an antibody that is selected for specificity
for a particular protein, polypeptide, or peptide. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein, polypeptide, or peptide.
For example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies specifically immunoreactive with a
protein, polypeptide, or peptide. See Harlow & Lane, 1988,
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, for a description of immunoassay formats and conditions
that can be used to determine specific immunoreactivity.
[0078] Antibodies, including binding fragments and single chain
recombinant versions thereof, against the invention peptides are
raised by immunizing animals, e.g., with conjugates of the peptides
with carrier proteins as described above. It is understood that
antibodies raised against any of the invention peptides may bind
native or mutant APP. However, of special interest are antibodies
raised against any of the invention peptides that do not bind
native or mutant APP but that do bind the peptide of the
invention.
[0079] The peptide substrates of the present invention may be used
to prepare polyclonal and/or monoclonal antibodies using
conventional techniques. The intact invention peptide, optionally
coupled to a carrier molecule, may be injected into small
vertebrates, with monoclonal antibodies being produced by
well-known methods, as described in detail below. Antibodies so
produced will be useful for performing conventional immunoassays to
detect peptides of the invention in biological and other specimens.
Antibodies according to the present invention will bind to the
invention peptides with high affinity of at least 10.sup.6
M.sup.-1. Likewise, the cleavage products produced as a result of
the cleavage of the invention peptides by .beta.-secretase can also
be used as immunogens for the production of antibodies specific
thereto.
[0080] A recombinant invention peptide, or a synthetic version
thereof, may be injected into an animal capable of producing
antibodies. Either monoclonal or polyclonal antibodies can be
generated for subsequent use in immunoassays to measure the
presence and quantity of the peptide.
[0081] Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably an
invention peptide, an invention peptide coupled to an appropriate
carrier (e.g., GST, keyhole limpet hemanocyanin, etc.) or
incorporated into an immunization vector such as a recombinant
vaccinia virus (see, e.g., U.S. Pat. No. 4,722,848) is mixed with
an adjuvant and animals are immunized with the mixture. The
animal's immune response to the immunogen preparation is monitored
by taking test bleeds and determining the titer of reactivity to
the peptide of interest. When approximately high titers of antibody
to the immunogen are obtained, blood is collected from the animal
and antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the peptide is performed where
desired. See, e.g., Coligan, 1991, Current Protocols in Immunology
Wiley/Greene, NY; and Harlow & Lane, 1989, Antibodies: A
Laboratory Manual Cold Spring Harbor Press, NY.
[0082] Monoclonal antibodies may be prepared from cells secreting
the desired antibody. In some instances, it is desirable to prepare
monoclonal antibodies from particular mammalian hosts, such as
mice, rodents, primates, humans, etc. Description of techniques for
preparing such monoclonal antibodies are found in, e.g., Stites et
al., eds., Basic and Clinical Immunology, 4th ed., Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow & Lane, supra; Goding, 1986, Monoclonal Antibodies:
Principles and Practice (2d ed.) Academic Press, New York, N.Y.;
and Kohler & Milstein, 1975, Nature 256:495-497. This method
proceeds by injecting an animal with an immunogen. The animal is
then sacrificed and cells taken from its spleen, which are fused
with myeloma cells. The result is a hybrid cell or "hybridoma" that
is capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0083] Alternative methods of immortalization include
transformation with Epstein Barr Virus, oncogenes, or retroviruses,
or other methods known in the art. Colonies arising from single
immortalized cells are screened for production of antibodies of the
desired specificity and affinity for the antigen, and the yield of
the monoclonal antibodies produced by such cells is enhanced by
various techniques, including injection into the peritoneal cavity
of a vertebrate (preferably mammalian) host. The antibodies of the
present invention are used with or without modification, and
include chimeric antibodies such as humanized murine
antibodies.
[0084] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors. See, Huse et
al., 1989, Science 246:1275-1281; and Ward et al., 1989, Nature
341: 544-546.
[0085] Frequently, the peptides and antibodies will be labeled by
joining, either covalently or non covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are know and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionucleotides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,272,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced. See, Cabilly, U.S. Pat. No.
4,816,567; and Queen et al., 1989, Proc. Natl. Acad. Sci. USA
86:10029-10033.
[0086] A "label" is a composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, or chemical means. For
example, useful labels include 32p, fluorescent dyes,
electron-dense reagents, enzymes (e.g., as commonly used in an
ELISA), biotin, dioxigenin, or haptens and proteins for which
antisera or monoclonal antibodies are available. A label often
generates a measurable signal, such as radioactivity, fluorescent
light or enzyme activity, which can be used to quantitate the
amount of bound label.
[0087] Labels can be attached directly or via spacer arms of
various lengths (to reduce steric hindrance). Any of a wide variety
of labeled reagents can be used for purposes of the present
invention. For instance, one or more labeled nucleoside
triphosphates, primers, linkers, or probes can be used. A
description of immunofluorescent analytic techniques is found in
DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool,
Marchalonis et al., eds., John Wiley & Sons, Ltd., pp. 189-231
(1982), which is incorporated herein by reference.
[0088] The term label can also refer to a "tag", which can bind
specifically to a labeled molecule. For instance, one can use
biotin as a tag and then use avidinylated or streptavidinylated
horseradish peroxidase (HRP) to bind to the tag, and then use a
chromogenic substrate (e.g., tetramethylbenzamine) to detect the
presence of HRP. In a similar fashion, the tag can be an epitope or
antigen (e.g., digoxigenin), and an enzymatically, fluorescently,
or radioactively labeled antibody can be used to bind to the
tag.
[0089] The antibodies of this invention are also used for affinity
chromatography in isolating the invention peptides. Columns are
prepared, e.g., with the antibodies linked to a solid support,
e.g., particles, such as agarose, Sephadex, or the like, where a
sample suspected of containing the invention peptides or even
native APP is passed through the column, washed and treated with
increased concentrations of a mild denaturant, whereby invention
peptides or the subject APP proteins are released.
[0090] The antibodies can be used to screen expression libraries
for particular expression products such as mammalian APP,
preferably bound to .beta.-secretase. Usually the antibodies in
such a procedure are labeled with a moiety allowing easy detection
of presence of antigen by antibody binding.
[0091] An alternative approach is the generation of humanized
immunoglobulins by linking the CDR regions of the non-human
antibodies to human constant regions by recombinant means. The
humanized immunoglobulins have variable region framework residues
substantially from a human immunoglobulin (termed an acceptor
immunoglobulin) and complementarity determining regions
substantially from a mouse immunoglobulin (referred to as the donor
immunoglobulin). The constant region(s), if present, are also
substantially from a human immunoglobulin. The human variable
domains are usually chosen from human antibodies whose framework
sequences exhibit a high degree of sequence identity with the
murine variable region domains from which the CDRs are derived. The
heavy and light chain variable region framework residues can be
derived from the same or different human antibody sequences. The
human antibody sequences can be the sequences of naturally
occurring human antibodies or can be consensus sequences of several
human antibodies. See International Patent Publication WO 92/22653.
Certain amino acids from the human variable region framework
residues are selected for substitution based on their possible
influence on CDR conformation and/or binding to antigen.
Investigation of such possible influences is by modeling,
examination of the characteristics of the amino acids at particular
locations, or empirical observation of the effects of substitution
or mutagenesis of particular amino acids.
[0092] Other candidates for substitution are acceptor human
framework amino acids that are unusual for a human immunoglobulin
at that position. These amino acids can be substituted with amino
acids from the equivalent position of the antibody or from the
equivalent positions of more typical human immunoglobulins.
[0093] A further approach for isolating DNA sequences which encode
a human monoclonal antibody or a binding fragment thereof is by
screening a DNA library from human B cells according to the general
protocol outlined by Huse et al., 1989, Science 246:1275-1281 and
then cloning and amplifying the sequences which encode the antibody
(or binding fragment) of the desired specificity. The protocol
described by Huse is rendered more efficient in combination with
phage display technology. See, e.g., International Patent
Publication WO 91/17271 and International Patent Publication WO
92/01047. Phage display technology can also be used to mutagenize
CDR regions of antibodies previously shown to have affinity for
.beta.-secretase protein receptors or their ligands. Antibodies
having improved binding affinity are selected.
[0094] An aspect of the invention provides methods for identifying
compounds which diminish or abolish interaction of .beta.-secretase
with its native substrate. The invention peptides may be employed
in a competitive binding assay. Such an assay can accommodate the
rapid screening of a large number of compounds to determine which
compounds, if any, are capable of inhibiting the binding of human
.beta.-secretase to a substrate. Subsequently, more detailed assays
can be carried out with those compounds found to bind, to further
determine whether such compounds act as inhibitors of human
.beta.-secretase cleavage activity.
[0095] As understood by those of skill in the art, assay methods
for identifying compounds that modulate proteolytic activity of
.beta.-secretase generally require comparison to a control. One
type of control is a cell or culture that is treated substantially
the same as the test cell or test culture exposed to the compound,
with the distinction that the control cell or culture is not
exposed to the compound.
[0096] Alternatively, a control system may include a peptide
substrate modified from any of those disclosed herein which
effectively is NOT cleaved by human .beta.-secretase.
[0097] Another type of control cell or culture may be a cell or
culture that is identical to the transfected cells, with the
exception that the control cell or culture do not express the
invention peptide. Accordingly, the response of the transfected
cell to compound is compared to the response (or lack thereof) of
the control cell or culture to the same compound under the same
reaction conditions.
[0098] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those prone to
have the disorder or those in which the disorder is to be
prevented.
[0099] By "screening" is meant investigating a collection of
substances (e.g., test compounds) for the presence or absence of a
property. Screening may include measuring or detecting various
properties, including the level of inhibition of .beta.-secretase
or the level of interaction between a .beta.-secretase and its
substrate.
[0100] By "therapeutically effective amount" is meant an amount of
a pharmaceutical composition that relieves to some extent one or
more symptoms of the disease or condition in the patient (e.g.,
prevents formation or additional deposit of amyloid plaques) or
returns to normal either partially or completely one or more
physiological or biochemical parameters associated with or
causative of the disease or condition.
[0101] An alternative embodiment of the invention provides a method
for treatment of Alzheimer's disease which comprises administering
to a patient an effective amount of a therapeutic compound which is
capable eof inhibiting the cleavage of native APP in said patient
and thereby preventing the increased deposition of amyloid plaque
in said patient.
[0102] Provided herein are nucleic acid molecules comprising a
sequence of nucleotides that encode any one of the herein disclosed
invention peptides. DNA sequences encoding any one or more of the
herein disclosed peptides can be used for recombinantly producing
the invention peptides when such nucleic acids are incorporated
into a variety of protein expression systems known to those of
skill in the art.
[0103] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and, unless otherwise limited, encompasses known analogs of natural
nucleotides that can function in a similar manner as naturally
occurring nucleotides. It will be understood that when a nucleic
acid molecule is said to have a DNA sequence, this also includes
RNA molecules having the corresponding RNA sequence in which "U"
replaces "T".
[0104] The term "recombinant host cell" refers to a cell having DNA
introduced from an exogenous source. Thus, for example, recombinant
host cells may express genes that are not found within the native
(non-recombinant) form of the cell or may express genes normally
found in the cell but which have been introduced into the cell in a
different manner, e.g., linked to different expression control
sequences.
[0105] Incorporation of cloned DNA into a suitable expression
vector, transfection of eukaryotic cells with a plasmid vector or a
combination of plasmid vectors, each encoding one or more distinct
genes or with linear DNA, and selection of transfected cells are
well known in the art (see, e.g., Sambrook et al., 1989, Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press). Suitable means for introducing (transducing)
expression vectors containing invention nucleic acid constructs
into host cells to produce transduced recombinant cells (i.e.,
cells containing recombinant heterologous nucleic acid) are
well-known in the art (see, for review, Friedmann, 1989, Science,
244:1275-1281; Mulligan, 1993, Science, 260:926-932, each of which
are incorporated herein by reference in their entirety).
[0106] Thus, an embodiment of the present invention provides
transformed host cells that recombinantly express the herein
disclosed invention peptides.
[0107] As used herein, a cell is said to be "altered to express a
desired peptide" when the cell, through genetic manipulation, is
made to produce a protein which it normally does not produce or
which the cell normally produces at lower levels. One skilled in
the art can readily adapt procedures for introducing and expressing
either genomic, cDNA, or synthetic sequences into either eukaryotic
or prokaryotic cells.
[0108] Provided by the present invention are isolated, purified,
and/or substantially pure peptide substrates (invention peptides)
which are listed in Table 1 and Table 2. Functionally equivalent
derivatives of the herein disclosed peptides are also included as
part of the invention. Such derivatives are those that have minor
substitutions in their amino acid sequences, but which are
nonetheless as effective as the native invention peptides from
which each was derived. Thus, functionally equivalent peptides have
the ability to act as substrates for .beta.-secretase.
[0109] "Invention peptide" or "invention peptide substrate" or
"peptide substrate sequences" or "peptide substrate" are used
interchangeably to refer to a non-naturally occurring peptide
comprising a contiguous sequence fragment of at least 8 amino
acids, and which comprises a synthetic .beta.-secretase cleavage
site, where the at least 8 amino acids have a sequence selected
from the group consisting of: SEQ ID NOs: 1-256, 258-263, and 264.
Fragments and homologs thereof are also intended to be encompassed
by the present invention.
[0110] The invention peptide substrates are characterized as
comprising a sequence of amino acids that is not only a suitable
substrate for .beta.-secretase cleavage but also is hydrolyzed at a
rate that is substantially faster than that attending a peptide
derived from the corresponding region of Swedish mutant APP (SEQ ID
NO: 257). Some of the substrates are cleaved at rates 33 times
faster than the Swedish mutant APP. One substrate, EVNFEVEF (SEQ ID
NO: 262), is cleaved at a rate 60 times faster than the Swedish
mutant APP.
[0111] Active invention peptide substrate analogs/fragments include
peptide analogs whose amino acid sequence differs from those of
either Table 1 or Table 2 by the inclusion of amino acid
substitutions, additions or deletions (e.g., active fragments).
Active fragments can be identified by empirically testing the
resulting analogs for activity.
[0112] Active analogs bearing substitutions can be prepared by
introducing selected amino acid substitutions into a peptide. Any
substitutions where the activity is maintained or enhanced will be
within the present invention, but usually the substitutions will be
"conservative" as defined herein. The number of substitutions is at
the discretion of the practitioner, but the amino acid sequence of
the resulting peptide must conform to the definition of active
peptide. Conservative amino acid substitutions refer to the
potential interchangeability of residues having similar side
chains. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains includes
serine and threonine; a group of amino acids having acidic side
chains includes aspartic acid and glutamic acid; a group of amino
acids having basic side chains includes lysine, arginine, and
histidine; and a group of amino acids having sulfur-containing side
chains includes cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
[0113] The invention peptides, biologically active fragments, and
functional equivalents thereof can be produced by chemical
synthesis. For example, synthetic peptides can be produced using
Applied Biosystems, Inc. Model 430A or 431A automatic peptide
synthesizer (Foster City, Calif.) employing the chemistry provided
by the manufacturer.
[0114] The invention peptides may also be in the form of fusion
polypeptides where the invention peptide is joined to all or a
portion of another protein. Fusions may be generated with
heterologous proteins (for example, a reporter polypeptide, a
binding polypeptide, or the like). Fusion polypeptides may be
formed either recombinantly or by synthetic methods which are
well-known in the art.
[0115] The peptides of the present invention may also have amino
acid residues which have been chemically modified by known
techniques, such as phosphorylation, sulfonation, biotinylation or
the addition of other moieties. In some embodiments, the
modifications will be useful for labeling reagents, purification
targets, affinity ligand targeting, or the like.
[0116] As used herein, substrate activity of human .beta.-secretase
refers to any activity characteristic of .beta.-secretase. Such
activity can typically be measured by one or more in vitro methods,
and frequently corresponds to an in vivo activity of the secretase
enzyme. Such activity may be measured by any method known to those
of skill in the art, such as, for example, assays that measure
liberation of cleavage products, e.g., N or C-terminal fragments of
the invention peptide substrates.
[0117] See Table 2 for a list of available invention peptide
substrates that are cleaved by human .beta.-secretase. Based upon
the sequences provided herein, additional substrates can be
identified and designed using known techniques.
[0118] The present invention further provides assays for detecting
.beta.-secretase mediated cleavage of peptide substrates such as
those exemplified in Table 2. The methods utilize a reaction system
which includes (i) a .beta.-secretase component and (ii) a
substrate component, preferably the invention peptide, where the
.beta.-secretase cleaves the substrate over time to produce
cleavage products. Thus, .beta.-secretase activity can be observed
and monitored over time as the amount of cleavage product(s)
increases. The amount of cleavage product(s) in the reaction system
can be measured in a variety of ways, including immunologic,
chromatographic, electrophoretic, and the like.
[0119] Such .beta.-secretase cleavage detection methods are
particularly useful for screening test compounds to determine their
ability to inhibit .beta.-secretase mediated cleavage of APP. In
such cases, a test compound is first identified by the methods
described herein using invention peptides. Those test compounds
that have the ability to inhibit the .beta.-secretase-mediated
cleavage of invention peptides are further tested for the ability
to inhibit .beta.-secretase-mediated cleavage of APP. The test
compound is added to a reaction system where the substrate
component is APP and the effect of the test compound on production
of cleavage product is observed. Those compounds which inhibit the
production of cleavage product(s) from APP are considered to be
potential therapeutic agents for treatment of conditions associated
with increased A.beta. production such as Alzheimer's disease.
[0120] The reaction system will usually comprise .beta.-secretase
that will be either a purified or partially purified native
.beta.-secretase obtained from a cellular source. The cellular
source may be a recombinant host cell that expresses
.beta.-secretase by virtue of having been transfected with an
expression vector encoding .beta.-secretase. Alternatively,
.beta.-secretase may be obtained from a cellular source that
naturally (i.e., non-recombinantly) expresses .beta.-secretase.
Such a non-recombinant source could be a cell line having a
sufficiently high level of expression of native .beta.-secretase.
The invention peptide substrate may include any one of the peptides
exemplified in Table 2. The peptide substrate may be recombinant or
synthetically derived. The reaction system can employ a wide
variety of solid phase detection systems which permit observance of
the production of .beta.-secretase cleavage products over time or
the disappearance of substrate over time. The methods will be
particularly useful for determining the ability of test compounds
to inhibit .beta.-secretase mediated cleavage.
[0121] The assay may be performed by combining an at least
partially purified .beta.-secretase with at least one invention
peptide substrate in the presence of the test substance. Conditions
are maintained such that the .beta.-secretase cleaves the invention
peptide substrate into an amino-terminal fragment and a
carboxy-terminal fragment in the absence of a substance which
inhibits such cleavage. Cleavage of the peptide substrate in the
presence of the test compound is compared with that in the absence
of the test compound, and those test substances which provide
significant inhibition of the cleavage activity (usually at least
about 25% inhibition, more usually at least about 50% inhibition,
preferably at least about 75% inhibition, and often at least about
90% inhibition or higher) are considered to be .beta.-secretase
inhibitors. Such .beta.-secretase inhibitors may then be subjected
to further in vitro and/or in vivo testing to determine if they
inhibit the production of A.beta. in cellular and animal models. As
well, the cleavage products thus produced can be purified and used
as immunogens to provide for antibodies specific for each of the
"amino terminal" and "carboxy terminal" fragments, which, in turn,
can be used to identify such fragments in other assays.
[0122] The screening assays of .beta.-secretase and the invention
peptide substrate are conveniently performed using "sandwich"
assays where the amino-terminal or the carboxy-terminal fragment
produced by cleavage is captured on a solid phase. The captured
fragment may then be detected using an antibody specific for the
end of the fragment exposed by .beta.-secretase cleavage. An
exemplary antibody is an antibody raised against any cleavage
products produced as a result of .beta.-secretase activity. The
binding of the antibody to the cleaved cleavage product is detected
using conventional labeling systems, such as horseradish peroxidase
or other detectable enzyme labels, which are bound to the antibody
directly (covalently), or indirectly through intermediate linking
substances, such as biotin and avidin.
[0123] The compounds selected above may also be used to inhibit
cleavage of native or recombinant APP by .beta.-secretase in an in
vitro assay. Compounds selected by the in vitro system, may in
turn, be used as listed below.
Pharmaceutical Compositions and Therapeutic Methods
[0124] The present invention further comprises methods for
inhibiting the .beta.-secretase mediated cleavage of APP to APP
cleavage products in cells, where the method comprises
administering to the cells compounds selected by the method
described herein. The compounds may be added to cell culture in
order to inhibit APP cleavage which results in A.beta. production.
The compounds may also be administered to a patient in order to
inhibit .beta.-secretase mediated APP cleavage which results in
pathogenic A.beta. production and the deposition of amyloid
.beta.-plaque associated with Alzheimer's Disease and other
A.beta.-related conditions.
[0125] The present invention further comprises pharmaceutical
compositions incorporating a compound selected by the
herein-described methods and including a pharmaceutically
acceptable carrier. Such pharmaceutical compositions should contain
a therapeutic or prophylactic amount of at least one compound
identified by the method of the present invention. The
pharmaceutically acceptable carrier can be any compatible,
non-toxic substance suitable to deliver the compounds to an
intended host. Sterile water, alcohol, fats, waxes, and inert
solids may be used as the carrier. Pharmaceutically acceptable
adjuvants, buffering agents, dispersing agents, and the like may
also be incorporated into the pharmaceutical compositions.
Preparation of pharmaceutical conditions incorporating active
agents is well described in the medical and scientific literature.
See, for example, Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa., 16th Ed., 1982, the disclosures of
which are incorporated herein by reference.
[0126] Frequently, it will be desirable or necessary to introduce
the pharmaceutical compositions directly or indirectly to the
brain. Direct techniques usually involve placement of a drug
delivery catheter into the host's ventricular system to bypass the
blood-brain barrier. Indirect techniques, which are generally
preferred, involve formulating the compositions to provide for drug
latentiation by the conversion of hydrophilic drugs into
lipid-soluble drugs. Latentiation is generally achieved through
blocking of the hydroxyl, carboxyl, and primary amine groups
present on the drug to render the drug more lipid-soluble and
amenable to transportation across the blood-brain barrier.
Alternatively, the delivery of hydrophilic drugs can be enhanced by
intra-arterial infusion of hypertonic solutions which can
transiently open the blood-brain barrier.
[0127] The concentration of the compound in the pharmaceutical
carrier may vary widely, i.e., from less than about 0.1% by weight
of the pharmaceutical composition to about 20% by weight, or
greater. Typical pharmaceutical composition for intramuscular
injection would be made up to contain, for example, one to four ml
of sterile buffered water and one .mu.g to one mg of a compound
identified by the methods of the present invention. A typical
composition for intravenous infusion could be made up to contain
100 to 500 ml of sterile Ringer's solution and about 1 to 100 mg of
the compound.
[0128] The pharmaceutical compositions of the present invention can
be administered for prophylactic and/or therapeutic treatment of
diseases related to the deposition of A.beta., such as Alzheimer's
disease, Down's syndrome, and advanced aging of the brain. In
therapeutic applications, the pharmaceutical compositions are
administered to a subject in need thereof already suffering from
the disease. The pharmaceutical compositions will be administered
in an amount sufficient to inhibit further deposition of A.beta.
plaque. An amount adequate to accomplish this is a "therapeutically
effective dose." Such a therapeutically effective dose will depend
on the extent of the disease, the size of the host, and the like,
but will generally range from about 1 .mu.g to 100 mg of the
compound per kilogram of body weight of the host, with dosages of
10 .mu.g to 1 mg/kg being more commonly employed.
[0129] For prophylactic applications, the pharmaceutical
compositions of the present invention are administered to a host
susceptible to the A.beta.-related disease, but not already
suffering from such disease. Such hosts may be identified by
genetic screening and clinical analysis, as described in the
medical literature (e.g. Goate, 1991, Nature 349:704-706). The
pharmaceutical compositions will be able to inhibit or prevent
deposition of the A.beta. plaque at a symptomatically early stage,
preferably preventing even the initial stages of the .beta.-amyloid
disease. The amount of the compound required for such prophylactic
treatment, referred to as a prophylactically-effective dosage, is
generally the same as described above for therapeutic
treatment.
.beta.-Secretase Substrates
[0130] The .beta.-secretase substrates of the invention resemble
the corresponding sequences from the Swedish mutation with specific
amino acid substitutions incorporated therein. The sequence from
the Swedish mutation corresponding to the peptides of the present
invention is shown in SEQ ID NO: 257. The Swedish mutation results
from a double substitution of ASN.sub.595-LEU.sub.596 for the
LYS.sub.595-MET.sub.596 which are present in the wild type 695
isoform of APP.
[0131] The invention peptides are characterized as not only
containing .beta.-secretase cleavage sites but also are hydrolyzed
faster than the Swedish mutant sequence, SEQ ID NO: 257. The herein
disclosed invention peptides can themselves provide substrates
containing a .beta.-secretase cleavage site and can serve as well
as a starting point for creating derivative peptides. A
distinguishing feature of the invention peptides is that data
disclosed herein show that some of the invention peptides are
hydrolyzed at least 33 times faster than the Swedish mutant APP
sequence of SEQ ID NO: 257.
[0132] Based on the disclosure provided herein .beta.-secretase
substrates can be produced using standard biochemical synthesis and
recombinant nucleic acid techniques. Techniques for chemical
synthesis of peptides are well known in the art. (See, for example,
Vincent, in Peptide and Protein Drug Delivery, New York, N.Y.,
Dekker, 1990).
[0133] Recombinant synthesis techniques for peptides are also well
known in the art. Such techniques employ a nucleic acid template
for peptide synthesis. Starting with a particular amino acid
sequence and the known degeneracy of the genetic code, a large
number of different encoding nucleic acid sequences can be
obtained. The degeneracy of the genetic code arises because almost
all amino acids are encoded for by different combinations of
nucleotide triplets or "codons". The translation of a particular
codon into a particular amino acid is well known in the art (see,
e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990).
[0134] Amino acids are encoded for by codons as follows:
TABLE-US-00001 A = Ala = Alanine: codons GCA, GCC, GCG, GCU C = Cys
= Cysteine: codons UGC, UGU D = Asp = Aspartic acid: codons GAC,
GAU E = Glu = Glutamic acid: codons GAA, GAG F = Phe =
Phenylalanine: codons UUC, UUU G = Gly = Glycine: codons GGA, GGC,
GGG, GGU H = His = Histidine: codons CAC, CAU J = Ile = Isoleucine:
codons AUA, AUC, AUU K = Lys = Lysine: codons AAA, AAG L = Leu =
Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU M = Met = Methionine:
codon AUG N = Asn = Asparagine: codons AAC, AAU P = Pro = Proline:
codons CCA, CCC, CCG, CCU Q = Gln = Glutamine: codons CAA, GAG R =
Arg = Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU S = Ser =
Serine: codons AGC, AGU, UCA, UCC, UCG, UCU T = Thr = Threonine:
codons ACA, ACC, ACG, ACU V = Val = Valine: codons GUA, GUC, GUG,
GUU W = Trp = Tryptophan: codon UGG Y = Tyr = Tyrosine: codons UAC,
UAU
[0135] Recombinant synthesis of peptides is achieved in a host cell
using an expression vector. An expression vector contains
recombinant nucleic acid encoding a desired peptide along with
regulatory elements for proper transcription and processing. The
regulatory elements that may be present include those naturally
associated with the recombinant nucleic acid and exogenous
regulatory elements not naturally associated with the recombinant
nucleic acid. Exogenous regulatory elements such as an exogenous
promoter can be useful for expressing recombinant nucleic acid in a
particular host.
[0136] Generally, the regulatory elements that are present in an
expression vector include a transcriptional promoter, a ribosome
binding site, a terminator, and an optionally present operator. A
preferred element is a polyadenylation signal providing for
processing in eukaryotic cells. Other preferred elements include an
origin of replication for autonomous replication in a host cell, a
selectable marker, a limited number of useful restriction enzyme
sites, and a potential for high copy number. Examples of expression
vectors are cloning vectors, modified cloning vectors, specifically
designed plasmids and viruses.
[0137] Nucleic acid encoding a peptide can be expressed in a cell
without the use of an expression vector employing, for example,
synthetic mRNA or native mRNA. Additionally, mRNA can be translated
in various cell-free systems such as wheat germ extracts and
reticulocyte extracts, as well as in cell based systems, such as
frog oocytes. Introduction of mRNA into cell based systems can be
achieved, for example, by microinjection.
[0138] Techniques for introducing nucleic acid into an appropriate
environment for expression, for expressing the nucleic acid to
produce protein, and for isolating expressed proteins are will
known in the art. Examples of such techniques are provided in
references such as Ausubel, Current Protocols in Molecular Biology,
John Wiley, 1987-1998, and Sambrook et al., in Molecular Cloning, A
Laboratory Manual, 2.sup.nd Edition, Cold Spring Harbor Laboratory
Press, 1989.
[0139] Examples are provided below to further illustrate different
features and advantages of the present invention. The examples also
illustrate useful methodology for practicing the invention. These
examples do not limit the claimed invention.
EXAMPLE 1
Synthesis of .beta.-Secretase Peptide Substrates of the
Invention
[0140] The present inventors have established the preferred
substrate amino acid sequence for .beta.-secretase, an enzyme
relevant to Alzheimer's Disease, through the utilization of
synthetic combinatorial peptide libraries based on the core
structure of amyloid precursor protein (APP) at the natural site of
cleavage by .beta.-secretase. A set of 8 synthetic peptide
libraries was generated such that each library included one amino
acid position along the APP polypeptide chain that was varied to
include each component of a set of amino acids.
[0141] These libraries were developed on an eight residue core
structure of APP that covered the known cleavage site of
.beta.-secretase, as shown in FIG. 1, where X is a set of all amino
acids, with the exception of Met and Cys. In each case, the library
was represented by an amino acid sequence where seven of the eight
residue positions were identical with the corresponding residues in
Swedish mutant APP and the eighth residue, indicated by X, was
varied through the set of amino acids, resulting in a library of
sequences. Thus, eight such libraries were developed by placing the
site of variation at each position along the APP amino acid
sequence. These libraries, as well as individual peptide
substrates, were obtained commercially or synthesized using a
standard peptide synthesis double-coupling protocol from Applied
Biosystems Incorporated (AB) and an AB peptide synthesizer (Foster
City, Calif.) model 430A. To produce a library of peptide
sequences, one equivalent of an equimolar mixture of a set of
Boc-protected amino acids was substituted in place of the residue
normally found at the respective position in Swedish mutant APP.
Consequently, 8 such libraries were generated by inserting the site
of amino acid mixture incorporation at each point along the APP
peptide chain from P4 to P4' as illustrated in FIG. 1. A general
report of the method of peptide library synthesis and
characterization can be found elsewhere. See Ramjit, H. G.; Kruppa,
G. H.; Spier, J. P.; Ross, III, C. W.; Garsky, V. M. "The
significance of monoisotopic and carbon-13 isobars for the
identification of a 19-component dodecapeptide library by positive
ion electrospray Fourier transform ion cyclotron resonance mass
spectrometry", Rapid Commun. Mass Spectrom. 2000, 14, 1368-1376,
which is incorporated by reference in its entirety.
EXAMPLE 2
Determination of .beta.-Secretase Activity
[0142] Following synthesis and purification, each of the eight
peptide libraries was incubated with the enzyme .beta.-secretase
and the relative rates of cleavage of each individual peptide
library component was followed with liquid chromatography/mass
spectrometry (LC/MS) with analysis of both the depletion of the
full length peptide substrate, as well as the formation of peptide
cleavage fragments. The enzyme .beta.-secretase was incubated at
concentrations ranging from 5 to 50 nM at pH 4.5 with each of the
peptide libraries and/or mixtures of synthetic peptide
substrates.
[0143] For all incubations, the concentration of each individual
peptide substrate was held at 1 .mu.M to avoid precipitation, which
could be observed at higher substrate concentrations with some of
the more insoluble substrates. The mixtures of substrates and
enzyme were incubated in an Agillent Technology 1100 series liquid
chromatograph with a temperature controlled autosampler. The
temperature for all .beta.-secretase enzymatic assays was held
constant at 37.degree. C. A 100 .mu.l aliquot of each reaction
mixture was injected onto a 1.times.50 mm C18 reverse phase Ic
column (Metachem Technologies Inc., Torrance, Calif.) every two
hours to monitor reaction progress. After injection, a highly
optimized solvent gradient was delivered to the column to maximize
resolving power for each of the substrate library components.
Optimal performance was obtained with a gradient starting at 20%
solvent B (B =Acetonitrile with 0.05% trifluoroacetic acid, A
=0.05% TFA in H20) and increasing to 40% solvent B over 40 minutes.
After the LC/MS run, the data were processed by selected-ion
chromatograms for each of the peptide substrate library components
by extracting 0.5 Da mass windows surrounding the calculated mass
values of each of the peptides.
[0144] Extracting these data at each reaction time allowed the
relative depletion of each substrate to be followed. For example,
FIG. 2 illustrates the differences in rates of cleavage observed
for all peptide library components from the P1' peptide library
when treated with the enzyme .beta.-secretase. Some peptide library
components were observed to react and the corresponding peak areas
decreased with time, while the peak areas of those that were not
processed by .beta.-secretase remained fairly constant. Those
components that were shown to deplete the most readily were
considered to be preferred residues at the site of variation. For
clarity, only the P1' library components that were most rapidly
hydrolyzed by .beta.-secretase (P1'=Glu and P1'=Ala), one
intermediate sequence (P1'=Asp--the residue normally present in
Swedish mutant APP), and two peptides that were not cleaved by
.beta.-secretase (P1'=Gly and P1'=Pro) are shown in FIG. 2. The
data predicted that either Glu or Ala in position P1' would be
superior substrates for the enzyme .beta.-secretase.
[0145] A similar relative preference for P1'=Glu or Ala was deduced
from the analysis of the formation of cleavage products, that is,
by the detection of peptide cleavage products representing the
C-terminal fragments from the P1' peptide library as shown in FIG.
3. In this case, the preferred residue leads to more rapid
formation of hydrolyzed product as shown by the more rapid and
comparable rates of formation of the products represented by
P1'=Ala and P1'=Glu. Again, the P1'=Asp product shows formation
consistent with this residue being less conducive to
.beta.-secretase hydrolysis than either Glu or Ala. Thus, the
preferred residues for the P1' substrate site were predicted to be
Glu and Ala, based on the P1' peptide library analysis.
[0146] A similar detailed analysis was carried out with each of the
8 peptide libraries from P4 to P4' and graph summarizing the
results of all peptide library work is shown in FIG. 4. In this
figure, the relative preference for each amino acid surveyed is
represented by the height of the bars at each position along the
peptide chain from P4 to P4'. TABLE-US-00002 TABLE 1 P4 P3 P2 P1
P1' P2' P3' P4' APP: E V N L D A E F Pre- D = E L\I\n F F A = E V V
= I/L/n P ferred 2nd I\L\n V Y I\L\n S I\L\n E W 3rd V N E Y I\L\n
A Y F
[0147] As shown in Table 1 above, the residues normally found in at
each position along the sequence of the Swedish version of APP were
only infrequently identified by this approach to be the most
preferred, or even the second most preferred residue. The data
allow the compilation of residues to generate substrate sequences
that are preferred by .beta.-secretase, or processed much more
rapidly than even the Swedish mutant sequence of APP. These
findings have been validated by designing and synthesizing specific
amino sequences incorporating the preferred amino acids for
residues at P1, P1' and P2'.
[0148] This new substrate sequence was shown to be hydrolyzed by
.beta.-secretase at least 30 times faster than the Swedish mutant
APP sequence. For example, FIG. 5 shows the results obtained with
individual, pure peptides obtained with sequence information
derived from the peptide library study. Five peptide sequences were
incubated simultaneously in the presence of .beta.-secretase to
determine if incorporation of the library results would result in a
superior substrate. This study was done to ascertain if
incorporation of the residues that were found to be preferred from
the peptide library studies would result in a substrate that is
hydrolyzed faster than the normal Swedish mutant APP sequence. Five
individual peptide sequences were incubated simultaneously with
.beta.-secretase, analogous to the peptide library experiments.
[0149] The peptide sequences used in these experiments were:
TABLE-US-00003 EVNLDAEF (SEQ ID NO: 257) EVNLAAEF (SEQ ID NO: 258)
EVNFAAEF (SEQ ID NO: 259) EVNChgAAEF (SEQ ID NO: 263) EVNChaAAEF
(SEQ ID NO: 264)
[0150] where Chg and Cha are cyclohexylglycine and
cyclohexylalanine residues, respectively. The non-natural Chg and
Cha were included to determine if variations on the aromatic ring
of F, found to be preferred for P1, would result in even faster
.beta.-secretase-mediated hydrolysis. As is shown in FIG. 5, the
single point mutation of the aspartic acid in position P1' to Ala
resulted in a substrate that was hydrolyzed by .beta.-secretase 10
times faster than the corresponding Swedish mutant sequence.
Additionally, inclusion of the predicted phenylalanine residue in
position P1 in conjunction with the Ala substitution at P1'
resulted in an additional factor of almost 2 increase in the
observed rate of hydrolysis with .beta.-secretase. This resulted in
a substrate that was hydrolyzed roughly 17 times faster than the
Swedish mutant sequence. Additional substitutions that have been
incorporated include the valine residue at P2' for a substrate that
has the sequence FAV at position P1-P1'-P2'. This FAV-containing
peptide was found be hydrolyzed by .beta.-secretase roughly 30
times faster than the Swedish mutant APP sequence. These results
validate the approach involving the establishment of a preferred
substrate sequence by combination of single point library study
results. Incorporation of additional residues to establish even
better is made possible by the present data. Table 2 discloses 256
sequences predicted to be good .beta.-secretase substrates from the
selection of the two most preferred residues at each position
through the 8-mer sequence. TABLE-US-00004 TABLE 2 SEQUENCE
SEQUENCE ID SEQUENCE SEQUENCE ID EIYLEVIW 1 EIYFEVVW 2 DIYLEVIW 3
DIYFEVVW 4 EVYLEVIW 5 EVYFEVVW 6 DVYLEVIW 7 DVFFEAIP 8 2EIFLEVIW 9
EIYLAAIP 10 DIFLEVIW 11 DIYLAAIP 12 EVFLEVIW 13 EVYLAAIP 14
DVFLEVIW 15 DVYLAAIP 16 EIYFEVIW 17 EIFLAAIP 18 DIYFEVIW 19
DIFLAAIP 20 EVYFEVIW 21 EVFLAAIP 22 DVYFEVIW 23 DVFLAAIP 24
EIFFEVIW 25 EIYFAAW 26 DIFFEVIW 27 DIYFAAIP 28 EVFFEVIW 29 EVYFAAIP
30 DVFFEVIW 31 DVYFAAIP 32 EIYLAVIW 33 EIFFAAIP 34 DIYLAVIW 35
DIFFAAIP 36 EVYLAVIW 37 EVFFAAIP 38 DVYLAVIW 39 DVFFAAIP 40
EIFLAVIW 41 EIYLEVVP 42 DIFLAVIW 43 DIYLEVVP 44 EVFLAVIW 45
EVYLEVVP 46 DVFLAVIW 47 DVYLEVVP 48 EIYFAVIW 49 EIFLEVVP 50
DIFFAAVW 51 DIFLEVVP 52 EVFFAAVW 53 EVFLEVVP 54 DVFFAAVW 55
DVFLEVVP 56 EIYLEVIP 57 DVYFEVVW 58 DIYLEVIP 59 EIFFEVVW 60
EVYLEVIP 61 DIFFEVVW 62 DVYLEVIP 63 EVFFEVVW 64 EIFLEVIP 65
DVFFEVVW 66 DIFLEVIP 67 EIYLAVVW 68 EVFLEVIP 69 DIYLAVVW 70
DVFLEVIP 71 EVYLAVVW 72 EIYFEVIP 73 DVYLAVVW 74 DIYFEVIP 75
EIFLAVVW 76 EVYFEVIP 77 DIFLAVVW 78 DVYFEVIP 79 EVFLAVVW 80
EIFFEVIP 81 DVFLAVVW 82 DIFFEVIP 83 EIYFAVVW 84 EVFFEVIP 85
DIYFAVVW 86 DVFFEVIP 87 EVYFAVVW 88 EIYLAVIP 89 DVYFAVVW 90
DIYLAVIP 91 EIFFAVVW 92 EVYLAVIP 93 DLFFAVVW 94 DVYLAVIP 95
EVFFAVVW 96 EIFLAVIP 97 DVFFAVVW 98 DIFLAVIP 99 EIYLEAVW 100
EVYFAAVP 101 DIYLEAVW 102 DVYFAAVP 103 EVYLEAVW 104 EIFFAAVP 105
DVYLEAVW 106 DIFFAAVP 107 EIYFEVVP 108 EVFFAAVP 109 DIYFEVVP 110
DVFFAAVP 111 EVYFEVVP 112 DIYFAVIW 113 DVYFEVVP 114 EVYFAVIW 115
EIFFEVVP 116 DVYFAVIW 117 DIFFEVVP 118 EIFFAVIW 119 EVFFEVVP 120
DIFFAVIW 121 DVFFEVVP 122 EVFFAVIW 123 EIYLAVVP 124 DVFFAVIW 125
DIYLAVVP 126 EIYLEAIW 127 EVYLAVVP 128 DIYLEAIW 129 DVYLAVVP 130
EVYLEAIW 131 EIFLAVVP 132 DVYLEAIW 133 DIFLAVVP 134 EIFLEAIW 135
EVFLAVVP 136 DIFLEAIW 137 DVFLAVVP 138 EVELFAIW 139 EIYFAVVP 140
DVFLEAIW 141 DIYFAVVP 142 EIYFEAIW 143 EVYFAVVP 144 DIYFEAIW 145
DVYFAVVP 146 EVYFEAIW 147 EIFFAVVP 148 DVYFEAIW 149 DLFFAVVP 150
EIFFEAIW 151 EVFFAVVP 152 DIFFEAIW 153 DVFFAVVP 154 EVFFEAIW 155
EIYLEAVP 156 DVFFEAIW 157 EIFLEAVW 158 EIYLAAIW 159 DIFLEAVW 160
DIYLAAIW 161 EVFLEAVW 162 EVFLAVIP 163 DVFLEAVW 164 DVFLAVIP 165
EIYFEAVW 166 EIYFAVIP 167 DIYFEAVW 168 DIYFAVIP 169 EVYFEAVW 170
EVYFAVIP 171 DVYFEAVW 172 DVYFAVIP 173 EIFFEAVW 174 EIFEAVIP 175
DIFFEAVW 176 DIFFAVIP 177 EVFFEAVW 178 EVEFAVIP 179 DVFFEAVW 180
DVFFAVIP 181 EIYLAAVW 182 ELYLEAIP 183 DIYLAAVW 184 DIYLEAIP 185
EVYLAAVW 186 EVYLEAIP 187 DVYLAAVW 188 DVYLEAIP 189 EIFLAAVW 190
EIFLEAIP 191 DIFLAAVW 192 DIFLEAIP 193 EVFLAAVW 194 EVFLEAIP 195
DVFLAAVW 196 DVFLEAIP 197 EIYFAAVW 198 EIYFEAIP 199 DIYFAAVW 200
DIYFEAIP 201 EVYFAAVW 202 EVYFEAIP 203 DVYFAAVW 204 DVYFEAIP 205
EIFFAAVW 206 EIFFEAIP 207 DIYLEAVP 208 DIFFEAIP 209 EVYLEAVP 210
EVFFEAIP 211 DVYLEAVP 212 EVYLAAIW 213 EIFLEAVP 214 DVYLAAIW 215
DIFLEAVP 216 EIFLAAIW 217 EVFLEAVP 218 DIFLAAIW 219 DVFLEAVP 220
EVFLAAIW 221 EIYFEAVP 222
DVFLAAIW 223 DIYFEAVP 224 EIYFAAIW 225 EVYFEAVP 226 DIYFAAIW 227
DVYFEAVP 228 EVYFAAIW 229 EIEFEAVP 230 DVYFAAIW 231 DIFFEAVP 232
EVYFAAIW 233 EVFFEAVP 234 DIFFAAIW 235 DVFFEAVP 236 EVFFAAIW 237
EIYLAAVP 238 DVFFAAIW 239 DIYLAAVP 240 EIYLEVVW 241 EVYLAAVP 242
DIYLEVVW 243 DVYLAAVP 244 EVYLEVYW 245 EIFLAAVP 246 DVYLEVVW 247
DIFLAAVP 248 EIFLEVVW 249 EVFLAAVP 250 DIYLEVVW 251 DVFLAAVP 252
EVFLEVVW 253 EIYFAAVP 254 DVFLEVVW 255 DIYFAAVP 256
EXAMPLE 3
Additional Assays
[0151] The .beta.-secretase peptide substrates can be employed in
assays measuring production of cleavage products. Cleavage of
.beta.-secretase substrates can be measured by detecting formation
of the N- or C-terminal cleavage products of any of the herein
disclosed peptide substrates. The presence of either of these
products can be measured using techniques such as those employing
antibodies and radioactive, electrochemiluminescent or fluorescent
labels. If needed or desirable, a purification step enriching the
different products may be employed. Examples of purification steps
include the use of antibodies, separation gels, and columns.
EXAMPLE 4
Cleavage Product Detection
[0152] The cleavage products of the herein disclosed peptide
substrates may be detected using a sandwich assay employing an
antibody to capture the cleavage peptide and an antibody to detect
the presence of the cleavage peptide. Detection may be achieved by
using electrochemiluminescence (ECL) (Yang et al., 1994,
Bio/Technology 12:193-194; Khorkova et al., 1998. Journal of
Neuroscience Methods 82:159-166), and an Origen 1.5 Analyzer (Igen
Inc., Gaithersburg, Md.).
EXAMPLE 5
Inhibition Studies
[0153] Inhibition studies can be performed to demonstrate that the
.beta.-secretase activity is catalyzed by a bona fide APP
processing enzyme in cells and is not simply due to a spurious
proteolytic activity. The studies may examine the effects of
various peptide substrates from Table 2 on cleavage at the
.beta.-secretase scissile bond of the substrates in an in vitro
assays using any known inhibitor of .beta.-secretase.
[0154] The effect of a .beta.-secretase inhibitor on
.beta.-secretase activity may, for example, be measured using
Chinese hamster ovary fibroblasts (CHO cells) that stably express
any one of the peptide substrates of the invention. CHO cells
expressing the invention peptides are grown in a suitable medium
under appropriate growth conditions, exemplified by 90% DMEM, 10%
fetal bovine serum, 2 mM glutamine, 100 .mu.g/ml each of penicillin
and streptomycin, and 0.2 mg/ml G418. The cells are seeded in
96-well dishes at 2.times.10.sup.4 cells/well. Cleavage product
formation may be detected using radiolabeled antibodies specific
for at least one cleavage product of the reference peptide
substrate that is used to transfect the CHO cells.
[0155] Treatment of CHO cells expressing the invention peptides
with a known .beta.-secretase inhibitor is expected to block
cleavage products from being secreted from such cells in a
dose-dependent manner with IC.sub.50 values for suppression of such
products that are similar to values reported in the literature.
[0156] Similarly, a .beta.-secretase inhibitor is expected to
inhibit "solubilized .beta.-secretase" mediated processing of any
of the invention peptide substrate sequences shown in Table 1 that
eventually result in the generation of the cleavage related
products.
EXAMPLE 6
A Preferred Substrate that is Hydrolyzed 60 Times Faster than the
Swedish Mutant
[0157] FIG. 6 shows the rate of hydrolysis observed for the Swedish
mutant (SEQ ID NO: 257) and four of the invention peptides. One
invention peptide in particular, EVNFEVEF (SEQ ID NO: 262), was
found to be an especially good subtrate for .beta.-secretase, with
its rate of hydrolysis being 60 times faster than the rate of
hydrolysis for SEQ ID NO: 257. Table 3 shows these results plus
results for EVNLAAEF (SEQ ID NO: 258). TABLE-US-00005 TABLE 3
Relative rates of hydrolysis EVNLDAEF (SEQ ID NO: 257) 1 EVNLAAEF
(SEQ ID NO: 258) 10 EVNFAAEF (SEQ ID NO: 259) 17 EVNFEAEF (SEQ ID
NO: 260) 17 EVNFAVEF (SEQ ID NO: 261) 30 EVNFEVEF (SEQ ID NO: 262)
60
[0158] Other embodiments are within the following claims. While
several embodiments have been shown and described, various
modifications may be made without departing from the spirit and
scope of the present invention.
Sequence CWU 1
1
264 1 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 1 Glu
Ile Tyr Leu Glu Val Ile Trp 1 5 2 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 2 Glu Ile Tyr Phe Glu Val Val Trp 1 5
3 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 3 Asp Ile
Tyr Leu Glu Val Ile Trp 1 5 4 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 4 Asp Ile Tyr Phe Glu Val Val Trp 1 5
5 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 5 Glu Val
Tyr Leu Glu Val Ile Trp 1 5 6 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 6 Glu Val Tyr Phe Glu Val Val Trp 1 5
7 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 7 Asp Val
Tyr Leu Glu Val Ile Trp 1 5 8 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 8 Asp Val Phe Phe Glu Ala Ile Pro 1 5
9 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 9 Glu Ile
Phe Leu Glu Val Ile Trp 1 5 10 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 10 Glu Ile Tyr Leu Ala Ala Ile Pro 1 5
11 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 11 Asp
Ile Phe Leu Glu Val Ile Trp 1 5 12 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 12 Asp Ile Tyr Leu Ala Ala Ile Pro 1 5
13 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 13 Glu
Val Phe Leu Glu Val Ile Trp 1 5 14 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 14 Glu Val Tyr Leu Ala Ala Ile Pro 1 5
15 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 15 Asp
Val Phe Leu Glu Val Ile Trp 1 5 16 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 16 Asp Val Tyr Leu Ala Ala Ile Pro 1 5
17 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 17 Glu
Ile Tyr Phe Glu Val Ile Trp 1 5 18 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 18 Glu Ile Phe Leu Ala Ala Ile Pro 1 5
19 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 19 Asp
Ile Tyr Phe Glu Val Ile Trp 1 5 20 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 20 Asp Ile Phe Leu Ala Ala Ile Pro 1 5
21 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 21 Glu
Val Tyr Phe Glu Val Ile Trp 1 5 22 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 22 Glu Val Phe Leu Ala Ala Ile Pro 1 5
23 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 23 Asp
Val Tyr Phe Glu Val Ile Trp 1 5 24 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 24 Asp Val Phe Leu Ala Ala Ile Pro 1 5
25 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 25 Glu
Ile Phe Phe Glu Val Ile Trp 1 5 26 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 26 Glu Ile Tyr Phe Ala Ala Ile Pro 1 5
27 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 27 Asp
Ile Phe Phe Glu Val Ile Trp 1 5 28 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 28 Asp Ile Tyr Phe Ala Ala Ile Pro 1 5
29 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 29 Glu
Val Phe Phe Glu Val Ile Trp 1 5 30 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 30 Glu Val Tyr Phe Ala Ala Ile Pro 1 5
31 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 31 Asp
Val Phe Phe Glu Val Ile Trp 1 5 32 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 32 Asp Val Tyr Phe Ala Ala Ile Pro 1 5
33 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 33 Glu
Ile Tyr Leu Ala Val Ile Trp 1 5 34 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 34 Glu Ile Phe Phe Ala Ala Ile Pro 1 5
35 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 35 Asp
Ile Tyr Leu Ala Val Ile Trp 1 5 36 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 36 Asp Ile Phe Phe Ala Ala Ile Pro 1 5
37 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 37 Glu
Val Tyr Leu Ala Val Ile Trp 1 5 38 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 38 Glu Val Phe Phe Ala Ala Ile Pro 1 5
39 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 39 Asp
Val Tyr Leu Ala Val Ile Trp 1 5 40 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 40 Asp Val Phe Phe Ala Ala Ile Pro 1 5
41 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 41 Glu
Ile Phe Leu Ala Val Ile Trp 1 5 42 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 42 Glu Ile Tyr Leu Glu Val Val Pro 1 5
43 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 43 Asp
Ile Phe Leu Ala Val Ile Trp 1 5 44 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 44 Asp Ile Tyr Leu Glu Val Val Pro 1 5
45 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 45 Glu
Val Phe Leu Ala Val Ile Trp 1 5 46 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 46 Glu Val Tyr Leu Glu Val Val Pro 1 5
47 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 47 Asp
Val Phe Leu Ala Val Ile Trp 1 5 48 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 48 Asp Val Tyr Leu Glu Val Val Pro 1 5
49 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 49 Glu
Ile Tyr Phe Ala Val Ile Trp 1 5 50 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 50 Glu Ile Phe Leu Glu Val Val Pro 1 5
51 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 51 Asp
Ile Phe Phe Ala Ala Val Trp 1 5 52 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 52 Asp Ile Phe Leu Glu Val Val Pro 1 5
53 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 53 Glu
Val Phe Phe Ala Ala Val Trp 1 5 54 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 54 Glu Val Phe Leu Glu Val Val Pro 1 5
55 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 55 Asp
Val Phe Phe Ala Ala Val Trp 1 5 56 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 56 Asp Val Phe Leu Glu Val Val Pro 1 5
57 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 57 Glu
Ile Tyr Leu Glu Val Ile Pro 1 5 58 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 58 Asp Val Tyr Phe Glu Val Val Trp 1 5
59 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 59 Asp
Ile Tyr Leu Glu Val Ile Pro 1 5 60 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 60 Glu Ile Phe Phe Glu Val Val Trp 1 5
61 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 61 Glu
Val Tyr Leu Glu Val Ile Pro 1 5 62 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 62 Asp Ile Phe Phe Glu Val Val Trp 1 5
63 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 63 Asp
Val Tyr Leu Glu Val Ile Pro 1 5 64 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 64 Glu Val Phe Phe Glu Val Val Trp 1 5
65 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 65 Glu
Ile Phe Leu Glu Val Ile Pro 1 5 66 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 66 Asp Val Phe Phe Glu Val Val Trp 1 5
67 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 67 Asp
Ile Phe Leu Glu Val Ile Pro 1 5 68 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 68 Glu Ile Tyr Leu Ala Val Val Trp 1 5
69 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 69 Glu
Val Phe Leu Glu Val Ile Pro 1 5 70 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 70 Asp Ile Tyr Leu Ala Val Val Trp 1 5
71 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 71 Asp
Val Phe Leu Glu Val Ile Pro 1 5 72 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 72 Glu Val Tyr Leu Ala Val Val Trp 1 5
73 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 73 Glu
Ile Tyr Phe Glu Val Ile Pro 1 5 74 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 74 Asp Val Tyr Leu Ala Val Val Trp 1 5
75 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 75 Asp
Ile Tyr Phe Glu Val Ile Pro 1 5 76 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 76 Glu Ile Phe Leu Ala Val Val Trp 1 5
77 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 77 Glu
Val Tyr Phe Glu Val Ile Pro 1 5 78 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 78 Asp Ile Phe Leu Ala Val Val Trp 1 5
79 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 79 Asp
Val Tyr Phe Glu Val Ile Pro 1 5 80 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 80 Glu Val Phe Leu Ala Val Val Trp 1 5
81 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 81 Glu
Ile Phe Phe Glu Val Ile Pro 1 5 82 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 82 Asp Val Phe Leu Ala Val Val Trp 1 5
83 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 83 Asp
Ile Phe Phe Glu Val Ile Pro 1 5 84 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 84 Glu Ile Tyr Phe Ala Val Val Trp 1 5
85 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 85 Glu
Val Phe Phe Glu Val Ile Pro 1 5 86 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 86 Asp Ile Tyr Phe Ala Val Val Trp 1 5
87 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 87 Asp
Val Phe Phe Glu Val Ile Pro 1 5 88 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 88 Glu Val Tyr Phe Ala Val Val Trp 1 5
89 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 89 Glu
Ile Tyr Leu Ala Val Ile Pro 1 5 90 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 90 Asp Val Tyr Phe Ala Val Val Trp 1 5
91 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 91 Asp
Ile Tyr Leu Ala Val Ile Pro 1 5 92 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 92 Glu Ile Phe Phe Ala Val Val Trp 1 5
93 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 93 Glu
Val Tyr Leu Ala Val Ile Pro 1 5 94 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 94 Asp Ile Phe Phe Ala Val Val Trp 1 5
95 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 95 Asp
Val Tyr Leu Ala Val Ile Pro 1 5 96 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 96 Glu Val Phe Phe Ala Val Val Trp 1 5
97 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 97 Glu
Ile Phe Leu Ala Val Ile Pro 1 5 98 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 98 Asp Val Phe Phe Ala Val Val Trp 1 5
99 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 99 Asp
Ile Phe Leu Ala Val Ile Pro 1 5 100 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 100 Glu Ile Tyr Leu Glu Ala Val Trp 1
5 101 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 101
Glu Val Tyr Phe Ala Ala Val Pro 1 5 102 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 102 Asp Ile Tyr Leu Glu Ala Val Trp 1
5 103 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 103
Asp Val Tyr Phe Ala Ala Val Pro 1 5 104 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 104 Glu Val Tyr Leu Glu Ala Val Trp 1
5 105 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 105
Glu Ile Phe Phe Ala Ala Val Pro 1 5 106 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 106 Asp Val Tyr Leu Glu Ala Val Trp 1
5 107 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 107
Asp Ile Phe Phe Ala Ala Val Pro 1 5 108 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 108 Glu Ile Tyr Phe Glu Val Val Pro 1
5 109 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 109
Glu Val Phe Phe Ala Ala Val Pro 1 5 110 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 110 Asp Ile Tyr Phe Glu Val Val Pro 1
5 111 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 111
Asp Val Phe Phe Ala Ala Val Pro 1 5 112 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 112 Glu Val Tyr Phe Glu Val Val Pro 1
5 113 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 113
Asp Ile Tyr Phe Ala Val Ile Trp 1 5 114 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 114 Asp Val Tyr Phe Glu Val Val Pro 1
5 115 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 115
Glu Val Tyr Phe Ala Val Ile Trp 1 5 116 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 116 Glu Ile Phe Phe Glu Val Val Pro 1
5 117 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 117
Asp Val Tyr Phe Ala Val Ile Trp 1 5 118 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 118 Asp Ile Phe Phe Glu Val Val Pro 1
5 119 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 119
Glu Ile Phe Phe Ala Val Ile Trp 1 5 120 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 120 Glu Val Phe Phe Glu Val Val Pro 1
5 121 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 121
Asp Ile Phe Phe Ala Val Ile Trp 1 5 122 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 122 Asp Val Phe Phe Glu Val Val Pro 1
5 123 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 123
Glu Val Phe Phe Ala Val Ile Trp 1 5 124 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 124 Glu Ile Tyr Leu Ala Val Val Pro 1
5 125 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 125
Asp Val Phe Phe Ala Val Ile Trp 1 5 126 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 126 Asp Ile Tyr Leu Ala Val Val Pro 1
5 127 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 127
Glu Ile Tyr Leu Glu Ala Ile Trp 1 5 128 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 128 Glu Val Tyr Leu Ala Val Val Pro 1
5 129 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 129
Asp Ile Tyr Leu Glu Ala Ile Trp 1 5 130 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 130 Asp Val Tyr Leu Ala Val Val Pro 1
5 131 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 131
Glu Val Tyr Leu Glu Ala Ile Trp 1 5 132 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 132 Glu Ile Phe Leu Ala Val Val Pro 1
5 133 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 133
Asp Val Tyr Leu Glu Ala Ile Trp 1 5 134 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 134 Asp Ile Phe Leu Ala Val Val Pro 1
5 135 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 135
Glu Ile Phe Leu Glu Ala Ile Trp 1 5 136 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 136 Glu Val Phe Leu Ala Val Val Pro 1
5 137 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 137
Asp Ile Phe Leu Glu Ala Ile Trp 1 5 138 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 138 Asp Val Phe Leu Ala Val Val Pro 1
5 139 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 139
Glu Val Phe Leu Glu Ala Ile Trp 1 5 140 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 140 Glu Ile Tyr Phe Ala Val Val Pro 1
5 141 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 141
Asp Val Phe Leu Glu Ala Ile Trp 1 5 142 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 142 Asp Ile Tyr Phe Ala Val Val Pro 1
5 143 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 143
Glu Ile Tyr Phe Glu Ala Ile Trp 1 5 144 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 144 Glu Val Tyr Phe Ala Val Val Pro 1
5 145 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 145
Asp Ile Tyr Phe Glu Ala Ile Trp 1 5 146 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 146 Asp Val Tyr Phe Ala Val Val Pro 1
5 147 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 147
Glu Val Tyr Phe Glu Ala Ile Trp 1 5 148 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 148 Glu Ile Phe Phe Ala Val Val Pro 1
5 149 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 149
Asp Val Tyr Phe Glu Ala Ile Trp 1 5 150 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 150 Asp Ile Phe Phe Ala Val Val Pro 1
5 151 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 151
Glu Ile Phe Phe Glu Ala Ile Trp 1 5 152 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 152 Glu Val Phe Phe Ala Val Val Pro 1
5 153 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 153
Asp Ile Phe Phe Glu Ala Ile Trp 1 5 154 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 154 Asp Val Phe Phe Ala Val Val Pro 1
5 155 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 155
Glu Val Phe Phe Glu Ala Ile Trp 1
5 156 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 156
Glu Ile Tyr Leu Glu Ala Val Pro 1 5 157 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 157 Asp Val Phe Phe Glu Ala Ile Trp 1
5 158 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 158
Glu Ile Phe Leu Glu Ala Val Trp 1 5 159 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 159 Glu Ile Tyr Leu Ala Ala Ile Trp 1
5 160 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 160
Asp Ile Phe Leu Glu Ala Val Trp 1 5 161 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 161 Asp Ile Tyr Leu Ala Ala Ile Trp 1
5 162 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 162
Glu Val Phe Leu Glu Ala Val Trp 1 5 163 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 163 Glu Val Phe Leu Ala Val Ile Pro 1
5 164 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 164
Asp Val Phe Leu Glu Ala Val Trp 1 5 165 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 165 Asp Val Phe Leu Ala Val Ile Pro 1
5 166 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 166
Glu Ile Tyr Phe Glu Ala Val Trp 1 5 167 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 167 Glu Ile Tyr Phe Ala Val Ile Pro 1
5 168 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 168
Asp Ile Tyr Phe Glu Ala Val Trp 1 5 169 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 169 Asp Ile Tyr Phe Ala Val Ile Pro 1
5 170 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 170
Glu Val Tyr Phe Glu Ala Val Trp 1 5 171 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 171 Glu Val Tyr Phe Ala Val Ile Pro 1
5 172 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 172
Asp Val Tyr Phe Glu Ala Val Trp 1 5 173 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 173 Asp Val Tyr Phe Ala Val Ile Pro 1
5 174 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 174
Glu Ile Phe Phe Glu Ala Val Trp 1 5 175 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 175 Glu Ile Phe Phe Ala Val Ile Pro 1
5 176 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 176
Asp Ile Phe Phe Glu Ala Val Trp 1 5 177 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 177 Asp Ile Phe Phe Ala Val Ile Pro 1
5 178 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 178
Glu Val Phe Phe Glu Ala Val Trp 1 5 179 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 179 Glu Val Phe Phe Ala Val Ile Pro 1
5 180 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 180
Asp Val Phe Phe Glu Ala Val Trp 1 5 181 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 181 Asp Val Phe Phe Ala Val Ile Pro 1
5 182 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 182
Glu Ile Tyr Leu Ala Ala Val Trp 1 5 183 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 183 Glu Ile Tyr Leu Glu Ala Ile Pro 1
5 184 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 184
Asp Ile Tyr Leu Ala Ala Val Trp 1 5 185 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 185 Asp Ile Tyr Leu Glu Ala Ile Pro 1
5 186 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 186
Glu Val Tyr Leu Ala Ala Val Trp 1 5 187 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 187 Glu Val Tyr Leu Glu Ala Ile Pro 1
5 188 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 188
Asp Val Tyr Leu Ala Ala Val Trp 1 5 189 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 189 Asp Val Tyr Leu Glu Ala Ile Pro 1
5 190 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 190
Glu Ile Phe Leu Ala Ala Val Trp 1 5 191 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 191 Glu Ile Phe Leu Glu Ala Ile Pro 1
5 192 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 192
Asp Ile Phe Leu Ala Ala Val Trp 1 5 193 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 193 Asp Ile Phe Leu Glu Ala Ile Pro 1
5 194 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 194
Glu Val Phe Leu Ala Ala Val Trp 1 5 195 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 195 Glu Val Phe Leu Glu Ala Ile Pro 1
5 196 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 196
Asp Val Phe Leu Ala Ala Val Trp 1 5 197 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 197 Asp Val Phe Leu Glu Ala Ile Pro 1
5 198 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 198
Glu Ile Tyr Phe Ala Ala Val Trp 1 5 199 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 199 Glu Ile Tyr Phe Glu Ala Ile Pro 1
5 200 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 200
Asp Ile Tyr Phe Ala Ala Val Trp 1 5 201 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 201 Asp Ile Tyr Phe Glu Ala Ile Pro 1
5 202 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 202
Glu Val Tyr Phe Ala Ala Val Trp 1 5 203 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 203 Glu Val Tyr Phe Glu Ala Ile Pro 1
5 204 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 204
Asp Val Tyr Phe Ala Ala Val Trp 1 5 205 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 205 Asp Val Tyr Phe Glu Ala Ile Pro 1
5 206 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 206
Glu Ile Phe Phe Ala Ala Val Trp 1 5 207 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 207 Glu Ile Phe Phe Glu Ala Ile Pro 1
5 208 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 208
Asp Ile Tyr Leu Glu Ala Val Pro 1 5 209 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 209 Asp Ile Phe Phe Glu Ala Ile Pro 1
5 210 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 210
Glu Val Tyr Leu Glu Ala Val Pro 1 5 211 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 211 Glu Val Phe Phe Glu Ala Ile Pro 1
5 212 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 212
Asp Val Tyr Leu Glu Ala Val Pro 1 5 213 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 213 Glu Val Tyr Leu Ala Ala Ile Trp 1
5 214 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 214
Glu Ile Phe Leu Glu Ala Val Pro 1 5 215 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 215 Asp Val Tyr Leu Ala Ala Ile Trp 1
5 216 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 216
Asp Ile Phe Leu Glu Ala Val Pro 1 5 217 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 217 Glu Ile Phe Leu Ala Ala Ile Trp 1
5 218 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 218
Glu Val Phe Leu Glu Ala Val Pro 1 5 219 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 219 Asp Ile Phe Leu Ala Ala Ile Trp 1
5 220 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 220
Asp Val Phe Leu Glu Ala Val Pro 1 5 221 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 221 Glu Val Phe Leu Ala Ala Ile Trp 1
5 222 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 222
Glu Ile Tyr Phe Glu Ala Val Pro 1 5 223 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 223 Asp Val Phe Leu Ala Ala Ile Trp 1
5 224 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 224
Asp Ile Tyr Phe Glu Ala Val Pro 1 5 225 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 225 Glu Ile Tyr Phe Ala Ala Ile Trp 1
5 226 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 226
Glu Val Tyr Phe Glu Ala Val Pro 1 5 227 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 227 Asp Ile Tyr Phe Ala Ala Ile Trp 1
5 228 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 228
Asp Val Tyr Phe Glu Ala Val Pro 1 5 229 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 229 Glu Val Tyr Phe Ala Ala Ile Trp 1
5 230 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 230
Glu Ile Phe Phe Glu Ala Val Pro 1 5 231 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 231 Asp Val Tyr Phe Ala Ala Ile Trp 1
5 232 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 232
Asp Ile Phe Phe Glu Ala Val Pro 1 5 233 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 233 Glu Ile Phe Phe Ala Ala Ile Trp 1
5 234 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 234
Glu Val Phe Phe Glu Ala Val Pro 1 5 235 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 235 Asp Ile Phe Phe Ala Ala Ile Trp 1
5 236 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 236
Asp Val Phe Phe Glu Ala Val Pro 1 5 237 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 237 Glu Val Phe Phe Ala Ala Ile Trp 1
5 238 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 238
Glu Ile Tyr Leu Ala Ala Val Pro 1 5 239 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 239 Asp Val Phe Phe Ala Ala Ile Trp 1
5 240 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 240
Asp Ile Tyr Leu Ala Ala Val Pro 1 5 241 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 241 Glu Ile Tyr Leu Glu Val Val Trp 1
5 242 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 242
Glu Val Tyr Leu Ala Ala Val Pro 1 5 243 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 243 Asp Ile Tyr Leu Glu Val Val Trp 1
5 244 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 244
Asp Val Tyr Leu Ala Ala Val Pro 1 5 245 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 245 Glu Val Tyr Leu Glu Val Val Trp 1
5 246 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 246
Glu Ile Phe Leu Ala Ala Val Pro 1 5 247 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 247 Asp Val Tyr Leu Glu Val Val Trp 1
5 248 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 248
Asp Ile Phe Leu Ala Ala Val Pro 1 5 249 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 249 Glu Ile Phe Leu Glu Val Val Trp 1
5 250 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 250
Glu Val Phe Leu Ala Ala Val Pro 1 5 251 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 251 Asp Ile Phe Leu Glu Val Val Trp 1
5 252 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 252
Asp Val Phe Leu Ala Ala Val Pro 1 5 253 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 253 Glu Val Phe Leu Glu Val Val Trp 1
5 254 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 254
Glu Ile Tyr Phe Ala Ala Val Pro 1 5 255 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 255 Asp Val Phe Leu Glu Val Val Trp 1
5 256 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 256
Asp Ile Tyr Phe Ala Ala Val Pro 1 5 257 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 257 Glu Val Asn Leu Asp Ala Glu Phe 1
5 258 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 258
Glu Val Asn Leu Ala Ala Glu Phe 1 5 259 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 259 Glu Val Asn Phe Ala Ala Glu Phe 1
5 260 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 260
Glu Val Asn Phe Glu Ala Glu Phe 1 5 261 8 PRT Artificial Sequence
Beta-Secretase Cleavage Site 261 Glu Val Asn Phe Ala Val Glu Phe 1
5 262 8 PRT Artificial Sequence Beta-Secretase Cleavage Site 262
Glu Val Asn Phe Glu Val Glu Phe 1 5 263 8 PRT Artificial Sequence
VARIANT (1)...(8) Xaa = Cyclohexylglycine Beta-Secretase Cleavage
Site 263 Glu Val Asn Xaa Ala Ala Glu Phe 1 5 264 8 PRT Artificial
Sequence VARIANT (1)...(8) Xaa = Cyclohexylalanine Beta-Secretase
Cleavage Site 264 Glu Val Asn Xaa Ala Ala Glu Phe 1 5
* * * * *